WO2015152684A1 - Pharmaceutical composition for preventing and treating inflammatory disease or pharmaceutical composition for healing wound comprising ethyl acetate fraction of schizandra chinensis extract as active ingredient - Google Patents

Pharmaceutical composition for preventing and treating inflammatory disease or pharmaceutical composition for healing wound comprising ethyl acetate fraction of schizandra chinensis extract as active ingredient Download PDF

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Publication number
WO2015152684A1
WO2015152684A1 PCT/KR2015/003380 KR2015003380W WO2015152684A1 WO 2015152684 A1 WO2015152684 A1 WO 2015152684A1 KR 2015003380 W KR2015003380 W KR 2015003380W WO 2015152684 A1 WO2015152684 A1 WO 2015152684A1
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WIPO (PCT)
Prior art keywords
ethyl acetate
acetate fraction
chinensis extract
schisandra chinensis
pharmaceutical composition
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PCT/KR2015/003380
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French (fr)
Korean (ko)
Inventor
김영국
김형진
이성윤
남기환
문옥성
서영원
윤원기
이기훈
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한국생명공학연구원
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Publication of WO2015152684A1 publication Critical patent/WO2015152684A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/79—Schisandraceae (Schisandra family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • composition for the prevention and treatment of inflammatory diseases or pharmaceutical composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient
  • the present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases or a wound wound useful pharmaceutical composition comprising an ethyl acetate fraction of Schizandra chinensis BAALL. Extract as an active ingredient.
  • Inflammation is one of the defense responses of biological tissues to external stimuli. When the inflammatory reaction occurs, various inflammatory factors are produced, which causes clinical symptoms such as redness, fever, swelling, pain, and dysfunction. Cytokines are proteins that are involved in cell proliferation, differentiation, and activation by secreting from various types of cells in vivo and play an important role in immune and inflammatory reactions.
  • IL-6 is a cytokine of known importance in inflammatory disease autoimmune disease or cancer and performs various functions in various organs. IL-6 is known to be expressed in various cells and is involved in various life phenomena such as immune response, inflammation, hematopoietic process, cell proliferation and differentiation. It is mediated by two types of receptors of IL-6, namely the common receptor chain gpl30 of the IL-6R and IL-6 family, which are specific to IL-6. IL-6 first binds to IL-6R, and the complex of IL-6 and IL-6R in turn binds to two gpl30s, forms a receptor complex, and initiates signaling into cells (Hi rano et al. 1997).
  • JAK2 activated by transphosphorylation and disrupted with activated JAK2, tyrosine residues in receptor cytoplasmic domains7 ⁇ phosphorylation, SH2 or other phosphotyrosine binding mot if It acts as a docking site for STAT3 (signal transducers and activators of transcription 3) proteins.
  • STAT3 bound to the cytoplasmic region of the receptor is released from the receptor after phosphorylation by JAK2.
  • Activated STAT3 are bonded to each other in the cytoplasm homodimer (homodimer) or by interrogating after achieved a dimer (heterodimer) move into the nucleus (nucleus), the expression sequences of the target gene (recognit ion sequence) in the "combined transcription ( transcription) (Levy, DE et al., Nat Rev Mol Cell Biol, 2002, 3, 651-62, Darnell, JE, Jr, Science, 1997, 277, 1630-1635).
  • STAT3 acts as a key transcriptional regulator in the vital signal transduction of inflammatory reactions through the IL-6 pathway. . Inhibition of IL-6-induced signaling system for the treatment of inflammatory and autoimmune diseases is being actively studied.
  • anti-IL-6 R antibody The use of anti-IL-6 receptor antibodies is the best known.
  • Synovial cell growth inhibitor for rheumatoid arthritis using the anti-IL-6 R antibody International Patent Publication No. 96/011020
  • plasmacytosis hyperimmunoglobulinemia
  • anemia nephritis using an anti-IL-6 R antibody
  • Treatment of diseases caused by IL-6 products such as cachexia, rheumatoid arthritis, herdsman's disease and vasculitis has been known (International Publication No. 96/012503).
  • anti-IL-6R antibody is used for the treatment of sensitive T cell-related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity and atopic dermatitis (WO 98/042377) and systemic erythematoses.
  • sensitive T cell-related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity and atopic dermatitis (WO 98/042377) and systemic erythematoses.
  • Characteristics of Crohn's disease International Patent Publication No. 99/047170
  • Treatment of pancreatitis International Publication No. 00/010607
  • Treatment of psoriasis International Publication No. 02/034292
  • the anti-IL-6 R antibody may have an epitope which is a part recognized as a foreign protein when introduced into an individual, it may still be immunogenic when used as a therapeutic agent.
  • many studies have been made to develop therapeutic agents for diseases mediated by interleukin-6GL-6) and STAT3 using small molecule compounds which are not proteins recognized by the immune system. Is being done.
  • Schizandra extract is a natural resource
  • the fraction can be usefully used as a pharmaceutical composition for the prevention and treatment of inflammatory diseases.
  • the invention has been completed.
  • An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or a wound healing pharmaceutical composition comprising ethyl acetate fraction of Schizandra chinensis extract having STAT3 inhibitory activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention provides a dietary supplement for the prevention and improvement of inflammatory diseases comprising an ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention provides a cosmetic composition for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention also provides a method for treating inflammatory disease, comprising administering a pharmaceutically effective amount of ethyl acetate fraction of Schizandra chinensis extract to an individual suffering from an inflammatory disease.
  • the present invention also provides a method for preventing inflammatory disease, comprising administering to a subject an ethyl acetate fraction of a pharmaceutically effective amount of Schisandra chinensis extract.
  • the present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a pharmaceutical composition for the prevention and treatment of inflammatory diseases.
  • the present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a dietary supplement for the prevention and improvement of inflammatory diseases.
  • the present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a cosmetic composition for preventing and improving inflammatory diseases.
  • the present invention provides a pharmaceutical composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention provides a skin external preparation composition for wound healing, comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention provides a cosmetic composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present invention also provides a wound healing method comprising the step of treating the wound with an ethyl acetate fraction of the pharmaceutically effective amount of Schizandra chinensis extract.
  • the present invention also provides the use of an ethyl acetate fraction of Schizandra chinensis extract for use as a composition for wound healing.
  • the terms used in the present invention are explained.
  • prevention refers to any action that inhibits or delays the progression of a related disease by administration of a composition of the present invention.
  • treatment refers to any action by which administration of a composition of the present invention improves or beneficially alters the symptoms of a related disease.
  • the term "administration" means providing a subject of any composition of the invention in any suitable manner.
  • the term "inf la ⁇ at ion” is one of the defense responses of biological tissues to a certain stimulus, and complex lesions involving three kinds of tissue degeneration, circulatory disorder and exudation, and tissue proliferation. It is called. In addition, it is the expression of defense mechanisms against various forms of infection or irritants in metabolites in vivo, and various chemical mediators are involved in the mechanism of inflammation. Do. This is a local protective reaction caused by injury or destruction of tissue, which acts to destroy, weaken or shield both the injury causing material and the injured tissue.
  • This inflammation is characterized by the perforation of microvascular vessels, the leakage of blood components into the limpid space, the migration of white blood cells into inflammatory tissues, and usually accompanied by clinical symptoms such as erythema, edema, hyperalgesia and pain.
  • Inter leukin-6 is a type of cytokine involved in various biological activities such as cell proliferation, differentiation and maturation. It also induces differentiation and increases the production of antibodies by differentiating B cells into plasma cells. Interleukin-6 is a phosphoglyco protein that is secreted from a variety of cells, including T cells and B cells, as well as monocytes, fibroblasts, and keratinocytes. It acts on metabolism and inflammation.
  • the term "STAT3 (signal transducers and act ivators of transcr ipt ion 3) '' is a protein that is activated through phosphorylation when stimulated by cytokines or growth factors, activated STAT3 is homo Induces the expression of various target genes by migrating a dimer or a heterodimer to the forming nucleus and is involved in cell growth, apoptosis, and inflammatory reaction
  • HepG2 cells treated with ethyl acetate fraction of Schizandra chinensis extract As a result of confirming the expression level of p-STAT3 (Phospho-STAT3), the composition of the present invention has the effect of inhibiting the expression of P-STAT3 by IL-6, these results are the ethyl acetate fraction of Schizandra chinensis extract as an active ingredient
  • the composition containing can be used for the purpose of preventing and treating inflammatory diseases.
  • the present invention also provides a pharmaceutical composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • Ethyl acetate fraction of the Schizandra chinensis extract is preferably prepared by a manufacturing method comprising the following steps, but not limited thereto:
  • step 3 drying the filtered extract of step 2) under reduced pressure.
  • step 4) fractionation of the extract obtained in step 3) with water, nucleic acid, and ethyl acetate to obtain a fraction.
  • the Schizandra chinensis of step 1) is grown or commercially available. Can be used without limitation.
  • the extraction solvent of step 1) may use water, alcohol or a mixture thereof. As the alcohol, C1 to C2 lower alcohol may be used, and as the lower alcohol, ethanol or methane may be used.
  • the extraction method is preferably using hot water extraction, dipping extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, shaking extraction, Soxhlet extraction or reflux extraction, extraction of reflux angle extraction and ultrasonic extraction, etc.
  • Conventional extraction methods in the art can be used, such as methods using devices or adsorption resins including XAD and HP-20, and are preferably heated to reflux extraction or extraction at room temperature, but are not limited thereto.
  • the extraction solvent may be extracted by adding 1 to 200 times the amount of dried Schizandra chinensis, more preferably 1 to 50 times, but is not limited thereto.
  • the extraction temperature is preferably 4 ° C to 100 ° C, but is not limited thereto.
  • the extraction time is preferably 1 to 168 hours, more preferably 4 to 72 hours, but is not limited thereto.
  • the number of extraction is preferably 3 to 5 times, it is more preferable to extract three times, but is not limited thereto.
  • the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto.
  • the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but not always limited thereto.
  • the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto.
  • the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.
  • the ethyl acetate fraction of the Schizandra chinensis extract of step 4) is suspended after the Schizandra chinensis extract (50.5 g) obtained in step 3) in water, nucleic acid, ethyl acetate is added sequentially to separate each layer Then, the ethyl acetate layer is preferably concentrated under reduced pressure and dried to obtain an ethyl acetate fraction, but is not limited thereto.
  • the vacuum concentration is preferably a vacuum rotary evaporator, but is not limited thereto.
  • the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying, the drying of the phase or freeze drying, more preferably freeze drying, but is not limited thereto.
  • the extract of Schizandra chinensis may be extracted with water, C1 to C2 lower alcohol, or a combination solvent thereof, by quenching, heating, ultrasonic waves or reflux, but not always limited thereto.
  • the ethyl acetate fraction of the Schizandra chinensis extract may be to inhibit phosphorylation of STAT3, but is not limited thereto.
  • Ethyl acetate fraction of the Schizandra chinensis extract may be to inhibit the production of NO (Ni tric Oxide), but is not limited thereto.
  • the inflammatory disease is selected from the group consisting of edema, autoimmune disease, arthritis, peritonitis, psoriasis, asthma, multiple sclerosis, allergy, bronchial spasm, destructive bone disease, infectious disease, degenerative disease, necrotic disease and inflammatory periodontal disease It may be, but is not limited thereto.
  • p is the activated STAT3 p
  • the expression level of -STAT3 was performed by Western blot. As a result, the ethyl acetate fraction of Schizandra chinensis extract was concentration dependent. It was confirmed that the inhibition of STAT3 phosphorylation (Fig. 1).
  • Schizandra chinensis extract and ethyl acetate fraction of the present invention inhibits STAT3 and phosphorylation by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo, thereby preventing and treating inflammatory diseases. And it can be usefully used as an active ingredient of the wound healing composition.
  • the pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.
  • compositions according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. And in the form of sterile injectable solutions.
  • Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sudrose, solbi, manny, xylly, erythri, malty, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyridone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil Can be mentioned.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid form preparations may include at least one excipient such as starch, calcium carbonate, water, or the like. Prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple brothers, lubricants such as magnesium stearate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl acrylate can be used.
  • Suppository bases include wi tepsol, macrogol, tween 61, cacao butter, laurige and glycerogelatin.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage may be based on the patient's condition, weight, age, The range varies depending on sex, diet, excretion rate, severity of disease, drug type, administration time, administration method, administration period and administration period.
  • the daily dose is 0.0001 nig / kg in the amount of lyophilized extracts and fractions according to the invention. To 500 nig / kg, preferably 0.001 nig / kg to 100 nig / kg, and may be administered once to several times a day if necessary.
  • the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers.
  • the present invention provides a health food for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits the phosphorylation of STAT3 by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo. It can be usefully used as an active ingredient.
  • the health food of the present invention may be added with ethyl acetate fraction of Schizandra chinensis extract as it is, or may be used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • ethyl acetate fraction of Schizandra chinensis extract examples include meat, sausage bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, 3 ⁇ 4, ice cream, including various kinds of soups, drinks , Teas, drinks, alcoholic beverages and vitamin complexes, and includes all healthy foods in the general sense.
  • the health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages.
  • the natural carbohydrates include sugars such as glucose, monosaccharides such as fructose, malsaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. to be.
  • sweeteners natural sweeteners such as taumartin, stevia extract, Synthetic sweeteners such as saccharin and aspartame can be used.
  • the proportion of the natural carbohydrate is generally from about 0.01 to 0.04 g, preferably from about 0.02 to 0.03 g, per 100 compositions of the present invention.
  • the health food of the present invention is a variety of nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, It may contain an alcoholic agent such as alcohol and carbonated drinks. In addition it may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention also provides a cosmetic composition for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • a cosmetic composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits phosphorylation of STAT3 by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo, thereby preventing and improving inflammatory diseases. It can be usefully used as an active ingredient. In addition, it can be usefully used as an active ingredient of the cosmetic composition for wound healing.
  • the cosmetic composition may be, for example, an emulsion, suspension, microemulsion, microcapsules, fine granulocytes or dihydrate (liposomes), or non-vesicle vesicle dispersant obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, or water phase. It can be provided in the form of cream, skin, lotion, powder, ointment, spray or cone stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. ⁇ «3
  • the cosmetic composition in addition to the ethyl acetate fraction of the Schizandra chinensis extract of the present invention, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or It may contain auxiliaries commonly used in the cosmetic field, such as any other ingredients commonly used in cosmetics.
  • the fraction of the present invention may be added in an amount of 1 to 15% by weight, preferably 2 to 10% by weight, to the cosmetic composition which is usually contained. have.
  • the present invention also provides a method for treating inflammatory disease, comprising administering a pharmaceutically effective amount of ethyl acetate fraction of Schizandra chinensis extract to an individual suffering from an inflammatory disease.
  • the present invention also provides a method for preventing inflammatory disease, comprising administering to the subject an ethyl acetate fraction of a pharmaceutically effective amount of Schisandra chinensis extract.
  • the pharmaceutically effective amount refers to an amount sufficient to treat Jalhwan at a reasonable benefit or risk ratio applicable to medical treatment, which means the type, severity, sensitivity of the drug to the active drug, and administration time. And route of administration and rate of release, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.
  • Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits the phosphorylation of STAT3 by IL-6 in HepG2 cells and exhibits a potent effect on inflammatory diseases by inhibiting the degree of inflammation induced by zymosan A in vivo. Inflammatory disease example It can be usefully used in rooms and treatment methods.
  • the present invention provides a skin external preparation composition for wound healing, comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • ethyl acetate extract of Schizandra chinensis extract of the present invention is used as an external preparation for skin, it is additionally used for fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers and foaming agents. agent), fragrances, surfactants, water, di- or nonionic emulsifiers, fillers, metal-ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents And adjuvant commonly used in the field of dermatology, such as lipid vesicles or any other ingredients conventionally used for external preparations for skin.
  • the ingredients can be introduced in amounts generally used in the field of dermatology.
  • Ethyl acetate fraction of Schizandra chinensis extract according to the present invention unlike other fractions have the effect of inhibiting the activation of the STAT3 pathway induced by IL-6, pharmaceutical compositions or wounds for the prevention and treatment of various inflammatory diseases associated with the STAT3 pathway It can be usefully used as a pharmaceutical composition for healing.
  • FIG. 1 is a diagram showing the phosphorylation of STAT3 protein by IL-6 treatment when water fraction, nucleic acid fraction and ethyl acetate fraction of Schizandra chinensis extract are pretreated.
  • Figure 2 is a diagram showing the production of NO induced by LPS when the ethyl acetate fraction of Schizandra chinensis extract.
  • Figure 3 is the treatment of ethyl acetate fraction of Schizandra chinensis extract, lr
  • Figure 15 shows the size.
  • Figure 4 is a diagram showing the change in weight and experimental animals when treated with ethyl acetate fraction of Schizandra chinensis extract.
  • Figure 5 is a diagram showing the degree of edema relief of the sole when treating the ethyl acetate fraction of Schizandra chinensis extract.
  • FIG. 6 is a diagram showing the average difference between the thickness of the left foot and the right foot of the comparative group, Zymosan treatment group, ethylaceteart fraction treatment group of Schisandra chinensis extract.
  • Figure 7 is a photograph showing the degree of edema relief of the sole by treatment of ethyl acetate fraction of Schizandra chinensis extract.
  • lysate complete solution (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3V04, 2 mM EDTA, 1 mM PMSF, 20 mM leupeptin, 20 nig /
  • the cells were lysed using mi aprotonin, and then centrifuged (13,000xg, 15 minutes) to obtain the supernatant as lysate. Protein concentration was quantified using a Bio-Rad DC protein analysis kit, separated using a 10% SDS—PAGE gel, and then transferred to PVDF membraneCWeatran S, pore size 0.2 urn).
  • RAW 264.7 cells cultured by the method of Experimental Example 1 was pretreated with ethyl acetate fraction of the Schizandra chinensis extract obtained in Example 1 at various concentrations, and incubated for 18 hours by treatment with 100 ng / ml LPS after 1 hour.
  • the size of the knee joint was examined after treatment with the material as described above.
  • the ethyl acetate fraction of Schisandra chinensis extract was confirmed to inhibit the hypertrophy of the knee joint lymph nodes by zymosan A.
  • the foot thickness was measured daily after treating the material as described above.
  • the foot thickness was 18.7 26.7, 26.4 25.4 24.5 23.6 ⁇ , and treated with ethyl acetate fraction of Schisandra chinensis extract 18.5 24.8, 24.6 23. 1, 21.7, 21.7 mm I confirm that this was reduced (FIG. 5).
  • the average difference between the paw and right foot of the group treated with nothing, Zymosan A treated group, the group treated with ethyl acetate fraction of Zymosan A and Schisandra chinensis extract was 1.0, 29.5, 18.5 ⁇ (FIG. 6), The difference could be seen in the picture (Fig. 7).
  • the ethyl acetate fraction of Schizandra chinensis extract of the present invention unlike water and nucleic acid fraction, inhibits phosphorylation of STAT3 by IL-6 in HepG2 cells, and inflammation induced by zymosan A in vivo. By suppressing the degree, it can be usefully used as an active ingredient for the composition for preventing and treating inflammatory diseases or for wound healing.
  • the above ingredients were mixed and layered in an airtight cloth to prepare a powder.
  • 0.1-5.0 parts by weight of the ethyl acetate fraction of Schizandra chinensis extract of the present invention was added to soup and broth to prepare health-promoting meat products, noodles soup and broth.
  • Brown rice, barley, glutinous rice, yulmu was alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.
  • Black beans, black sesame seeds, and perilla were also roasted by a known method and then roasted to prepare a powder having a particle size of 60 mesh using a mill.
  • the ethyl acetate fraction of Schizandra chinensis extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and a hot air dryer, and then dried to a particle size of 60 mesh by a grinder. Grinding to obtain a dry powder.
  • the ethyl acetate fractions of the grains ⁇ seeds and Schisandra chinensis extracts prepared above were prepared by combining the following ratios.
  • Cereals (30 parts by weight brown rice, 15 parts by weight of radish, 20 parts by weight of barley) ,
  • Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),
  • Liquid fructose 0.5%), oligosaccharide (2), sugar (2%), salt (0.5%), combined homogeneously times the ethyl acetate fraction 5 g of the present "inventors Schizandra extract and the sub material, such as water (75%) After instant sterilization it was prepared by packaging in small packaging containers such as glass bottles and plastic bottles.
  • Vegetable juice was prepared by adding 5 g of ethyl acetate fraction of Schizandra chinensis extract to tomato or carrot juice 1,000 m £.
  • Fruit juice was prepared by adding 1 g of ethyl acetate fraction of Schizandra chinensis extract to 1,000 apple or grape juices.
  • Preparation Example 4 Preparation of Cosmetics
  • the present invention can be prepared cosmetics for the prevention and improvement of inflammatory diseases containing ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
  • the present inventors prepared cosmetics of solubilized formulations, such as cosmetics of the emulsified formulations such as nutrient cosmetics, creams, essences and soft cosmetics as the cosmetics.
  • step 3 While passing through the step 3), the mixture of 2) was slowly added to emulsify for 2-3 minutes at 6,000 rpm.
  • step 6) After quantifying the raw materials of 15 to 17, the mixture of step 5) was further emulsified at 40 ° C. for 30 seconds.
  • step 6) The emulsion of step 6) was emulsified and then degassed and cooled to 25-35 ° C. to prepare an emulsified cosmetic.
  • Emulsifier 1 Emulsifier 2 Emulsifier 3
  • a cosmetic of a solubilized formulation was prepared with the composition shown in Table 2 below.
  • the manufacturing method is as follows.
  • step 2) The mixture of step 2) was solubilized with slow addition to the mixture of step 1).

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating STAT3-mediated diseases comprising, as an active ingredient, a non-polar organic solvent extract of Schizandra chinensis having a STAT3 inhibitory activity. Since the Schizandra chinensis extract and the organic solvent extract thereof according to the present invention inhibit a STAT3 activity effectively, the extracts can be effectively used for the prevention and treatment of STAT3-mediated diseases such as inflammation.

Description

[명세세  [Specification
【발명의 명칭】  [Name of invention]
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질 환의 예방 및 치료용 약학적 조성물 또는 상처치유용 약학적 조성물  Pharmaceutical composition for the prevention and treatment of inflammatory diseases or pharmaceutical composition for wound healing, comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient
【기술분야】 Technical Field
본 발명은 오미자( Schizandra chinensis BAALL . ) 추출물의 에틸아세테이트 분획물 을 유효성분으로 포함하는 염증 질환의 예방 및 치료용 약학적 조성물 또는 상처치 유용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases or a wound wound useful pharmaceutical composition comprising an ethyl acetate fraction of Schizandra chinensis BAALL. Extract as an active ingredient.
. . .  . . .
[배경기술]  [Background]
염증은 외부 자극에 대한 생체조직의 방어반웅의 하나이며 염증반응이 일어 나면 여러 가지 염증 인자들이 만들어지고 이로 안하여 임상적으로는 발적， 발열， 종창， 동통， 기능장애 등의 증상이 나타낮다. 사이토카인은 생체 내 여러 종류의 세포에서 분비하여 세포 증식, 분화， 활성화 등에 관여하는 단백질로 면역 및 염증 반웅에 중요한 역할을 한다.  Inflammation is one of the defense responses of biological tissues to external stimuli. When the inflammatory reaction occurs, various inflammatory factors are produced, which causes clinical symptoms such as redness, fever, swelling, pain, and dysfunction. Cytokines are proteins that are involved in cell proliferation, differentiation, and activation by secreting from various types of cells in vivo and play an important role in immune and inflammatory reactions.
IL-6는 염증성 질환 자가 면역질환 또는 암에서 그 중요성이 알려진 사이 토카인으로 여러 장기에서 다양한 기능을 수행한다. IL-6는 다양한 세포에서 발현 됨이 알려져 있으며 , 면역반웅， 염증， 조혈과정， 세포의 증식 및 분화 등 다양한 생명 현상에 관여한다. IL-6의 2가지 종류의 수용체에 의해 매개되는데， 즉 IL-6 에 특이한 수용체인 IL-6R과 IL-6 패밀리의 공통수용 체인 gpl30이 이에 해당한다. IL-6은 먼저 IL-6R과 결합하게 되고, IL-6과 IL-6R의 복합체는 다시 2개의 gpl30과 결합， 수용체 복합체를 형성하여 세포 내로의 신호전달을 개시한다 (Hi rano 등 1997) . 야누스 키나제 2(Janus Kinases 2， JAK2)는 인산전이 (transphosphorylation)에 의하여 활성화되고, 활성화된 JAK2에 와해 수용체 세포 질 영역 (cytoplasmic domains)의 여러 티로신 잔기 (tyrosine residues )7} 인산화 (phosphorylation)되면， SH2나 다른 포스포티로신 바인딩 모티프 (phosphotyrosine binding mot if)를 가지고 있는 STAT3( signal transducers and activators of transcription 3) 단백질의 도킹사이트 (docking site) 역할을 하게 된다. 수용체 의 세포질 영역에 결합한 STAT3는 JAK2에 의해 인산화된 후 수용체에서 떨어져 나 온다. 활성화된 STAT3는 세포질 내에서 서로 결합하여 호모다이머 (homodimer) 또 는 해테로다이머 (heterodimer)를 이룬 후 핵 (nucleus)으로 이동， 타겟 유전자의 인 식 서열 (recognit ion sequence)에 '결합하여 전사 (transcription)를 조절한다 (Levy, DE et al. , , Nat Rev Mol Cell Biol, 2002, 3， 651-62, Darnell, J.E., J.r., Science, 1997, 277， 1630-1635). 이렇게 STAT3는 IL-6경로를 통한 염증 반웅의 생체 신호 전달에서 핵심 전사조절자로서 작용한다. . 염증성， 자가면역질환 등의 치료를 위해서 IL-6에 의해 유도되는 신호전달 체계의 억제에 대하여 연구가 활발히 진행되고 있으며， 현재 IL-6의 신호전달체계 의 억제에 대하여 항 IL-6 R 항체 (anti-IL-6 receptor antibody)를 이용한 것이 가 장 많이 알려져 있다. 상기 항 IL-6 R 항체를 이용한 류마티스성 관절염에 대한 활액 세포 성장 억제제 (국제특허공개 제 96/011020호)， 항 IL-6 R 항체를 이용 한 형질구 증가증， 초면역 글로불린 혈증， 빈혈， 신염ᅳ 악액질, 류마티스성 관절염， 목축업자 질병 및 맥관증식신염과 같은 IL-6 산물에 기인하는 질병의 치료 등이 이 미 알려진 바 있다 (국제특허공개공보 제 96/012503호). 또한， 항 IL-6 R항체는 다 발성 경화증， 포도막염， 만성 갑상선염， 지연성 과민증 및 아토피성 피부염과 같은 민감성 T 세포 관련 질병 (국제특허공개공보 제 98/042377호)， 전신성 에리테마토스 의 치료， 크론병의 차료 (국제특허공개공보제 99/047170호)ᅳ 췌장염의 치료 (국제특 허공개공보 제 00/010607호)， 건선의 치료 (국제특허공개공보 제 02/034292호) 및 연 소성 만성 관절염의 치료 (국제특허공개공보 제 02/080969호)에 적용될 수 있음이 보 고되었다. 그러나， 상기 항 IL-6 R 항체의 경우 개체 내에 도입되는 경우 외래 단 백질로서 인지될 수 있는 부분인 에피토프를 가질 수 있으므로， 치료제로서 사용될 경우 여전히 면역원성일 수 있다. 상기와 같은 문제점을 해결하기 위하여 면역체 계에 인지되지 않는 단백질이 아닌 작은 분자 화합물 (smal l molecule compound)을 이용하여 인터루킨 -6GL-6) 및 STAT3에 의해 매개되는 질병의 치료제를 개발하기 위한 많은 연구가 이루어지고 있다. 이에 본 발명자들은， 상기와 같이 다수의 염증성 질환 또는 자가 면역질환 의 병리에 있어서 증요한 역할을 하는 IL-6에 의한 신호전달과정을 조절할 수 있는 염증치료제를 탐색하는 과정에서， 천연자원인 오미자 추출물의 에틸아세테이트 분 획물이 농도 의존적으로 STAT3 활성을 억제하고 부종의 경감에 효과가 있음을 확인 함으로써ᅳ 상기 분획물을 염증 관련 질환의 예방 및 치료용 약학적 조성물로 유용 하게 사용할 수 있음을 밝힘으로써， 본 발명을 완성하였다. IL-6 is a cytokine of known importance in inflammatory disease autoimmune disease or cancer and performs various functions in various organs. IL-6 is known to be expressed in various cells and is involved in various life phenomena such as immune response, inflammation, hematopoietic process, cell proliferation and differentiation. It is mediated by two types of receptors of IL-6, namely the common receptor chain gpl30 of the IL-6R and IL-6 family, which are specific to IL-6. IL-6 first binds to IL-6R, and the complex of IL-6 and IL-6R in turn binds to two gpl30s, forms a receptor complex, and initiates signaling into cells (Hi rano et al. 1997). Janus Kinases 2, JAK2 activated by transphosphorylation and disrupted with activated JAK2, tyrosine residues in receptor cytoplasmic domains7} phosphorylation, SH2 or other phosphotyrosine binding mot if It acts as a docking site for STAT3 (signal transducers and activators of transcription 3) proteins. STAT3 bound to the cytoplasmic region of the receptor is released from the receptor after phosphorylation by JAK2. Activated STAT3 are bonded to each other in the cytoplasm homodimer (homodimer) or by interrogating after achieved a dimer (heterodimer) move into the nucleus (nucleus), the expression sequences of the target gene (recognit ion sequence) in the "combined transcription ( transcription) (Levy, DE et al., Nat Rev Mol Cell Biol, 2002, 3, 651-62, Darnell, JE, Jr, Science, 1997, 277, 1630-1635). Thus STAT3 acts as a key transcriptional regulator in the vital signal transduction of inflammatory reactions through the IL-6 pathway. . Inhibition of IL-6-induced signaling system for the treatment of inflammatory and autoimmune diseases is being actively studied. Currently, anti-IL-6 R antibody ( The use of anti-IL-6 receptor antibodies is the best known. Synovial cell growth inhibitor for rheumatoid arthritis using the anti-IL-6 R antibody (International Patent Publication No. 96/011020), plasmacytosis, hyperimmunoglobulinemia, anemia, nephritis using an anti-IL-6 R antibody Treatment of diseases caused by IL-6 products such as cachexia, rheumatoid arthritis, herdsman's disease and vasculitis has been known (International Publication No. 96/012503). In addition, anti-IL-6R antibody is used for the treatment of sensitive T cell-related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity and atopic dermatitis (WO 98/042377) and systemic erythematoses. ， Characteristics of Crohn's disease (International Patent Publication No. 99/047170) ᅳ Treatment of pancreatitis (International Publication No. 00/010607), Treatment of psoriasis (International Publication No. 02/034292) It has been reported that it can be applied to the treatment of plastic chronic arthritis (International Patent Publication No. 02/080969). However, since the anti-IL-6 R antibody may have an epitope which is a part recognized as a foreign protein when introduced into an individual, it may still be immunogenic when used as a therapeutic agent. In order to solve the above problems, many studies have been made to develop therapeutic agents for diseases mediated by interleukin-6GL-6) and STAT3 using small molecule compounds which are not proteins recognized by the immune system. Is being done. Accordingly, the present inventors, as described above, in the process of searching for an inflammatory therapeutic agent that can regulate the signaling process by IL-6, which plays an important role in the pathology of a number of inflammatory diseases or autoimmune diseases, Schizandra extract is a natural resource By confirming that the ethyl acetate fraction of ethanol has a concentration-dependent effect on inhibiting STAT3 activity and reducing edema, the fraction can be usefully used as a pharmaceutical composition for the prevention and treatment of inflammatory diseases. The invention has been completed.
【발명의 상세한 설명】 [Detailed Description of the Invention]
[기술적 과제】  [Technical Challenges]
본 발명의 목적은 STAT3 억제활성을 가지는 오미자 추출물의 에틸아세테이 트 분획물을 유효성분으로 포함하는 염증 질환의 예방 및 치료용 약학적 조성물 또 는 상처치유용 약학적 조성물을 제공하는 것이다.  An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or a wound healing pharmaceutical composition comprising ethyl acetate fraction of Schizandra chinensis extract having STAT3 inhibitory activity as an active ingredient.
[기술적 해결방법】 [Technical Solution]
상기 목적을 달성하기 위하여， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질환의 예방 및 치료용 약학적 조성물을 제 공한다. 또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질환의 예방 및 개선용 건강 기능 식품을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient. In addition, the present invention provides a dietary supplement for the prevention and improvement of inflammatory diseases comprising an ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한， 본 발명은 오미자 추출물의 에틸아세테이트 분확물을 유효성분으로 포함하는 염증 질환의 예방 및 개선용 화장료 조성물을 제공한다.  In addition, the present invention provides a cosmetic composition for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한， 본 발명은 약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 염증 질환에 걸린 개체에 투여하는 단계를 포함하는 염증 질환 치료방법 을 제공한다. - 또한， 본 발명은 약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 개체에 투여하는 단계를 포함하는 염증 질환 예방방법을 제공한다.  The present invention also provides a method for treating inflammatory disease, comprising administering a pharmaceutically effective amount of ethyl acetate fraction of Schizandra chinensis extract to an individual suffering from an inflammatory disease. The present invention also provides a method for preventing inflammatory disease, comprising administering to a subject an ethyl acetate fraction of a pharmaceutically effective amount of Schisandra chinensis extract.
또한， 본 발명은 염증 질환 예방 및 치료용 약학적 조성물로 사용하기 위한 오미자 추출물의 에틸아세테이트 분획물의 용도를 제공한다.  The present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a pharmaceutical composition for the prevention and treatment of inflammatory diseases.
또한， 본 발명은 염증 질환 예방 및 개선용 건강 기능 식품으로 사용하기 위한 오미자 추출물의 에틸아세테이트 분획물의 용도를 제공한다.  The present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a dietary supplement for the prevention and improvement of inflammatory diseases.
또한， 본 발명은 염증 질환 예방 및 개선용 화장료 조성물로 사용하기 위한 오미자 추출물의 에틸아세테이트 분획물의 용도를 제공한다.  The present invention also provides the use of ethyl acetate fraction of Schizandra chinensis extract for use as a cosmetic composition for preventing and improving inflammatory diseases.
또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 약학적 조성물을 제공한다.  In addition, the present invention provides a pharmaceutical composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 피부외용제 조성물을 제공한다.  In addition, the present invention provides a skin external preparation composition for wound healing, comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 화장료 조성물을 제공한다 .  In addition, the present invention provides a cosmetic composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한， 본 발명은 약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 상처에 처리하는 단계를 포함하는 상처치유 방법을쎄공한다.  The present invention also provides a wound healing method comprising the step of treating the wound with an ethyl acetate fraction of the pharmaceutically effective amount of Schizandra chinensis extract.
또한， 본 발명은 상처치유용 조성물로 사용하기 위한 오미자 추출물의 에틸 아세테이트 분획물의 용도를 제공한다. 본 발명에서 사용되는 용어를 설명한다. The present invention also provides the use of an ethyl acetate fraction of Schizandra chinensis extract for use as a composition for wound healing. The terms used in the present invention are explained.
본 발명에서 사용되는 용어 "예방 "은 본 발명의 조성물의 투여로 관련질환 을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다.  As used herein, the term "prevention" refers to any action that inhibits or delays the progression of a related disease by administration of a composition of the present invention.
본 발명에서 사용되는 용어 "치료" 또는 "개선''은 본 발명의 조성물의 투여 로 관련질환의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.  As used herein, the term "treatment" or "improvement" refers to any action by which administration of a composition of the present invention improves or beneficially alters the symptoms of a related disease.
본 발명에서 사용되는 용어 "투여 "는 임의의 적절한 방법으로 개체에 소정 의 본 발명의 조성물을 제공하는 것을 의미한다.  As used herein, the term "administration" means providing a subject of any composition of the invention in any suitable manner.
본 발명에서 용어， "염증 ( inf la醒 at ion) "이란 어떤 자극에 대한 생체조직의 방어반웅의 하나로， 조직 변질， 순환장애와 삼출 (出)， 조직 증식의 세 가지를 병발 하는 복잡한 병변을 일컫는다. 또한， 여러 가지 형태의 감염 ( infect ion)이나 생체 내 대사산물 중의 자극성 물질에 대한 생체 내 방어기전의 발현이라 할 수 있고， 다양한 화학적 매개체가 염증의 발현 기전에 관여하고 있으며， 그 병인도 매우 복 잡하다. 이는 조직의 상해 또는 파괴에 의해 유발되는 국소 보호 반웅으로， 상해 유발 물질과 상해된 조직 모두를 파괴， 약화시키거나 차폐하는 작용을 한다. 이러 한 염증의 특징은 미세혈관이 천공되고， 혈액 성분이 름새 공간으로 누출되며， 백 혈구가 염증 조직으로 이동한다는 것으로， 통상적으로 홍반， 부종， 통각 과민 및 통증 등의 임상적 증상들을 동반한다.  In the present invention, the term "inf la 醒 at ion" is one of the defense responses of biological tissues to a certain stimulus, and complex lesions involving three kinds of tissue degeneration, circulatory disorder and exudation, and tissue proliferation. It is called. In addition, it is the expression of defense mechanisms against various forms of infection or irritants in metabolites in vivo, and various chemical mediators are involved in the mechanism of inflammation. Do. This is a local protective reaction caused by injury or destruction of tissue, which acts to destroy, weaken or shield both the injury causing material and the injured tissue. This inflammation is characterized by the perforation of microvascular vessels, the leakage of blood components into the limpid space, the migration of white blood cells into inflammatory tissues, and usually accompanied by clinical symptoms such as erythema, edema, hyperalgesia and pain.
' 본 발명에서 용어， "인터루킨 -6( inter leukin-6 ; IL-6) "은 세포의 증식， 분 화， 성숙 같은 다양한 생물학적 활성에 관여하는 사이토카인의 한 종류로 면역반응 과 관련된 B세포와분화를 유도하기도 하고, B세포를 플라즈마 세포로 분화시켜 항 체 생성량을 증가시키는 역할을 한다. 인터루킨 -6는 인당단백질 (phosphoglyco protein)로서 T세포， B세포 뿐만 아니라 단핵세포 (monocyte)， 섬유아세포 ( f ibrobl ast ) 및 각질형상 세포 (kerat inocyte) 등 다양한 세포로부터 분비되며 생 체내의 면역과 대사， 염증발생에 작용한다. 본 발명에서 용어, "STAT3( signal transducers and act ivators of transcr ipt ion 3) ' '는 사이토카인이나 성장인자 (growth factor )에 의해 자극시， 인 산화를 통해 활성화되는 단백질로써 , 활성화된 STAT3는 호모다이머 또는 헤테로다 이머를 형성 핵으로 이동하여 다양한 타겟 유전자의 발현을 유도함으로써 세포성장 이나사멸, 염증반웅에 관여한다. 본 발명의 실시예에서는 오미자추출물의 에틸아 세테이트 분획물을 처리한 HepG2 세포에서， p-STAT3(Phospho-STAT3)의 발현 수준을 확인한 결과 본 발명의 조성물이 IL-6에 의한 P-STAT3의 발현을 억제하는 효과가 있으며， 이러한 결과는 오미자추출물의 에틸아세테이트 분획물올 유효성분으로 포 함하는 조성물이 염증 질환을 예방 및 치료시킬 수 있는 용도로 사용할 수 있음을 의미한다 . 이하, 본 발명을 상세히 설명한다. 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하 는 염증 질환의 예방 및 치료용 약학적 조성물을 제공한다. In the present invention, the term "inter leukin-6 (IL-6)" is a type of cytokine involved in various biological activities such as cell proliferation, differentiation and maturation. It also induces differentiation and increases the production of antibodies by differentiating B cells into plasma cells. Interleukin-6 is a phosphoglyco protein that is secreted from a variety of cells, including T cells and B cells, as well as monocytes, fibroblasts, and keratinocytes. It acts on metabolism and inflammation. In the present invention, the term "STAT3 (signal transducers and act ivators of transcr ipt ion 3) '' is a protein that is activated through phosphorylation when stimulated by cytokines or growth factors, activated STAT3 is homo Induces the expression of various target genes by migrating a dimer or a heterodimer to the forming nucleus and is involved in cell growth, apoptosis, and inflammatory reaction In an embodiment of the present invention, HepG2 cells treated with ethyl acetate fraction of Schizandra chinensis extract ， As a result of confirming the expression level of p-STAT3 (Phospho-STAT3), the composition of the present invention has the effect of inhibiting the expression of P-STAT3 by IL-6, these results are the ethyl acetate fraction of Schizandra chinensis extract as an active ingredient It means that the composition containing can be used for the purpose of preventing and treating inflammatory diseases. Ming provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
또한 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 약학적 조성물을 제공한다.  The present invention also provides a pharmaceutical composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
상기 오미자 추출물의 에틸아세테이트 분획물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나， 이에 한정되지 않는다 :  Ethyl acetate fraction of the Schizandra chinensis extract is preferably prepared by a manufacturing method comprising the following steps, but not limited thereto:
1) 오미자에 추출용매를 가하여 추출하는 단계;  1) extracting by adding an extraction solvent to the Schizandra chinensis;
2) 단계 1)의 추출물을 여과하는 단계 ; 및  2) filtering the extract of step 1); And
3) 단계 2)의 여과한 추출물을 감압농축한 후 건조하는 단계.  3) drying the filtered extract of step 2) under reduced pressure.
4) 단계 3)에서 수득한 추출물을 물， 핵산, 및 에틸 아세테이트로 분획하여 분획물을 얻는 단계  4) fractionation of the extract obtained in step 3) with water, nucleic acid, and ethyl acetate to obtain a fraction.
상기 방법에 있어서， 단계 1)의 오미자는 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 오미자 추출물은 오미자 열매를 이용하였다. 상기 방법에 있어서， 상기 단계 1)의 상기 추출용매는 물， 알코을 또는 이 들의 흔합물을 사용할 수 있다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용 할수 있으며， 상기 저급 알코을로는 에탄올 또는 메탄을을 이용할 수 있다. In the above method, the Schizandra chinensis of step 1) is grown or commercially available. Can be used without limitation. The Schisandra chinensis extract used Schisandra chinensis fruit. In the method, the extraction solvent of step 1) may use water, alcohol or a mixture thereof. As the alcohol, C1 to C2 lower alcohol may be used, and as the lower alcohol, ethanol or methane may be used.
상기 추출방법으로는 열수 추출， 침지 추출， 초임계 추출, 아임계 추출, 고 온 추출， 고압 추출， 진탕 추출， Soxhlet 추출 또는 환류 추출올 이용하는 것이 바 람직하고， 환류 넁각 추출 및 초음파 추출 등의 추출장치를 이용한 방법 또는 XAD 및 HP-20을 포함한 흡착 수지를 이용하는 방법 등 당업계의 통상적인 추출방법올 사용할 수 있으며， 가온하여 환류 추출 또는 상온에서 추출하는 것이 바람직하나， 이에 한정되지 않는다.  The extraction method is preferably using hot water extraction, dipping extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, shaking extraction, Soxhlet extraction or reflux extraction, extraction of reflux angle extraction and ultrasonic extraction, etc. Conventional extraction methods in the art can be used, such as methods using devices or adsorption resins including XAD and HP-20, and are preferably heated to reflux extraction or extraction at room temperature, but are not limited thereto.
상기 추출용매를 건조된 오미자 분량에 1 내지 200배 첨가하여 추출할 수 있고， 1 내지 50배로 하는 것이 더욱 바람직하나， 이에 한정되지 않는다. 상기 추 출온도는 4°C 내지 100°C인 것이 바람직하나 이에 한정하지 않는다.  The extraction solvent may be extracted by adding 1 to 200 times the amount of dried Schizandra chinensis, more preferably 1 to 50 times, but is not limited thereto. The extraction temperature is preferably 4 ° C to 100 ° C, but is not limited thereto.
또한， 상기 추출시간은 1 내지 168시간인 것이 바람작하며， 4 내지 72시간 이 더욱 바람직하나 , 이에 한정하지 않는다 .  In addition, the extraction time is preferably 1 to 168 hours, more preferably 4 to 72 hours, but is not limited thereto.
아울러， 추출 회수는 3 내지 5회인 것이 바람직하며， 3회 반복 추출하는 것 이 더욱 바람직하나 이에 한정되는 것은 아니다.  In addition, the number of extraction is preferably 3 to 5 times, it is more preferable to extract three times, but is not limited thereto.
상기 방법에 있어서， 단계 3)의 감압농축은 진공감압농축기 또는 진공회전 증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건 조， 진공건조， 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정 하지 않는다.  In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but not always limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전 증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한， 건조는 감압건 조, 진공건조， 비등건조， 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정 하지 않는다. 구체적으로， 단계 4)의 오미자 추출물의 상기 에틸아세테이트 분획물은 상 기 단계 3)에서 얻은 오미자 추출물 (50.5 g)을 물에 현탁시킨 후， 핵산， 에틸아세 테이트를 순차적으로 첨가하여 각각의 층을 분리한 다음 에틸아세테이트 층을 감압 농축 및 건조시켜 에틸아세테이트 분획물을 수득하는 것이 바람직하나， 이에 한정 되지 않는다. In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto. Specifically, the ethyl acetate fraction of the Schizandra chinensis extract of step 4) is suspended after the Schizandra chinensis extract (50.5 g) obtained in step 3) in water, nucleic acid, ethyl acetate is added sequentially to separate each layer Then, the ethyl acetate layer is preferably concentrated under reduced pressure and dried to obtain an ethyl acetate fraction, but is not limited thereto.
이때， 상기 감압 농축은 진공회전증발기를 이용하는 것이 바람직하나, 이에 한정되지 않는다.  At this time, the vacuum concentration is preferably a vacuum rotary evaporator, but is not limited thereto.
또한， 상기 건조는 감압 건조, 진공 건조， 비등 건조， 분무 건조, 상은 건 조 또는 동결 건조를 하는 것이 바람직하고 동결 건조를 하는 것이 더욱 바람직하 나， 이에 한정되지 않는다.  In addition, the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying, the drying of the phase or freeze drying, more preferably freeze drying, but is not limited thereto.
상기 오미자 추출물은 오미자를 물， C1내지 C2의 저급 알콜 또는 이들의 흔 합용매를 이용하여 넁침， 가열， 초음파 또는 환류 넁각으로 추출한 것일 수 있으나， 이에 한정되지 않는다.  The extract of Schizandra chinensis may be extracted with water, C1 to C2 lower alcohol, or a combination solvent thereof, by quenching, heating, ultrasonic waves or reflux, but not always limited thereto.
상기 오미자 추출물의 에틸아세테이트 분획물은 STAT3의 인산화를 억제하는 것일 수 있으나， 이에 한정되지 않는다.  The ethyl acetate fraction of the Schizandra chinensis extract may be to inhibit phosphorylation of STAT3, but is not limited thereto.
상기 오미자 추출물의 에틸아세테이트 분획물은 N0(Ni tr ic Oxide)의 생성을 억제하는 것일 수 있으나， 이에 한정되지 않는다.  Ethyl acetate fraction of the Schizandra chinensis extract may be to inhibit the production of NO (Ni tric Oxide), but is not limited thereto.
상기 염증성 질환은 부종， 자가면역질환， 관절염， 복막염, 건선， 천식， 다 발성경화증， 알레르기， 기관지 경련， 파괴성 골질환， 감염성 질환， 퇴행성 질환, 괴사성 질환 및 염증성 치주질환으로 구성된 군으로부터 선택되는 것일 수 있으나, 이에 한정되지 않는다.  The inflammatory disease is selected from the group consisting of edema, autoimmune disease, arthritis, peritonitis, psoriasis, asthma, multiple sclerosis, allergy, bronchial spasm, destructive bone disease, infectious disease, degenerative disease, necrotic disease and inflammatory periodontal disease It may be, but is not limited thereto.
본 발명의 구체적인 실시예에서, 오미자 추출물이 간암세포주 (HepG2)에서 IL-6 처리에 의한 STAT3 활성화에 미치는 영향을 확인하기 위하여， 오미자 추출물 의 각 분획물을 HepG2에 처리한 후， 활성화된 STAT3인 p-STAT3의 발현량을 웨스턴 블롯으로 수행하였고， 그 결과 오미자추출물의 에틸아세테이트 분획물이 농도의존 적으로 STAT3의 인산화를 억제함을 확인하였다 (도 1) . In a specific embodiment of the present invention, in order to determine the effect of Schisandra chinensis extract on STAT3 activation by IL-6 treatment in liver cancer cell line (HepG2), after treatment of each fraction of Schisandra chinensis extract to HepG2, p is the activated STAT3 p The expression level of -STAT3 was performed by Western blot. As a result, the ethyl acetate fraction of Schizandra chinensis extract was concentration dependent. It was confirmed that the inhibition of STAT3 phosphorylation (Fig. 1).
또한， 오미자추출물의 에틸아세테이트 분획물이 염증반웅의 중요세포인 대 식세포 (macrophage)에 미치는 영향을 살펴보기 위하여， 생쥐의 RAW 264.7 대식세포 에 LPS( l ipopolysacchar ide)를 처리하여 염증 정도를 나타내는 NO의 양을 측정한 결과， 오미자의 에틸아세테이트 분획물이 NO의 생성을 억제함을 확인하였다 (도 2) . in vivo에서 오미자추출물의 에틸아세테이트 분획물이 염증성 질환에 대하 여 치료효과를 가지는지 알아보기 위하여， zymosan A를 복강내에 투여하여， 염증 을 유도한 후 슬관절 림프절의 크기를 조사하였다. 그 결과， 오미자추출물의 에틸 아세테이트 분획물을 처리한 경우 zymosan A를 단독으로/처리한 군에 비해 림프절 이 비대해진 정도가 작음을 확인하였다 (도 3) . 본 발명의 분획물 처리에 의한 체 중감소는 없었는 바 부작용이 없음을 확인하였다 (도 4) . 또한， zymosan A를 오른 쪽 발에 주사하여 염증을 유도하고， 발 두께를 측정한 결과， 오미자추출물의 에틸 아세테이트 분획물을 처리한 경우 zymosan A만 처리한 군에 비해 발의 두께를 유의 하게 감소시키는 것을 확인하였다 (도 5， 도 6， 도 7) . 이는 오미자추출물의 에틸 아세테이트 분획물이 세포 실험결과에 상웅하게 in vivo에서 또한 염증 경감 활성 이 있음을 보여준다. .  In addition, to investigate the effect of ethyl acetate fraction of Schizandra chinensis extract on macrophage, an important cell of inflammatory reactions, LPS (l ipopolysacchar ide) was applied to RAW 264.7 macrophages in mice. As a result of measuring the amount, it was confirmed that ethyl acetate fraction of Schisandra chinensis inhibited the production of NO (FIG. 2). In order to investigate whether the ethyl acetate fraction of Schizandra chinensis extract in vitro has a therapeutic effect against inflammatory diseases, zymosan A was administered intraperitoneally to induce inflammation and then the size of knee joint lymph nodes was investigated. As a result, when the ethyl acetate fraction of Schizandra chinensis extract was treated, it was confirmed that the degree of lymph node enlargement was smaller than that of zymosan A alone / treatment group (FIG. 3). There was no weight loss by the fraction treatment of the present invention was confirmed that there are no side effects (Fig. 4). In addition, zymosan A was injected into the right foot to induce inflammation, and foot thickness was measured. When the ethyl acetate fraction of Schizandra chinensis extract was treated, it was confirmed that the foot thickness was significantly reduced compared to the group treated with zymosan A only. (FIGS. 5, 6, and 7). This shows that the ethyl acetate fraction of Schizandra chinensis extract also has anti-inflammatory activity in vivo, which is comparable to the cell results. .
따라서， 본 발명의 오미자 추출물와 에틸아세테이트 분획물은 HepG2 세포에 서 IL-6에 의한 STAT3와 인산화를 억제하고， in vivo에서 zymosan A에 의해 유도되 는 염증 정도를 억제함으로써, 염증 질환 예방 및 치료용 조성물 및 상처치유용 조 성물의 유효성분으로 유용하게 사용될 수 있다. 또한， 본 발명의 약학적 조성물은 약제의 제조에 통상적으로 사용하는 적절 한 담체， 부형제 및 희석제를 더 포함할 수 있다.  Thus, Schizandra chinensis extract and ethyl acetate fraction of the present invention inhibits STAT3 and phosphorylation by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo, thereby preventing and treating inflammatory diseases. And it can be usefully used as an active ingredient of the wound healing composition. In addition, the pharmaceutical composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.
본 발명에 따른 약학적 조성물은， 각각 통상의 방법에 따라 산제， 과립제， 정제, 캡슐제, 현탁액, 에멀젼， 시럽， 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학적 조성물 에 포함될 수 있는 담체， 부형제 및 희석제로는 락토즈， 덱스트로즈， 수 H로스， 솔 비를， 만니를， 자일리를， 에리스리를， 말티를， 전분, 아카시아 고무， 알지네이트， 젤라틴， 칼슘 포스페이트， 칼슘 실리케이트 셀를로즈， 메틸 셀를로즈， 미정질 셀 를로스， 폴리비닐 피를리돈， 물, 메틸히드록시벤조에이트, 프로필히드톡시벤조에이 트， 탈크， 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보 통 사용하는 충진제， 증량제, 결합제， 습윤제， 붕해제， 계면활성제 등의 희석제 또 는 부형제를 사용하며 조제된다. 경구투여를 위한 고형제제는 정제， 환제， 산제， 과립제， 캡슐제 등이 포함되며， 이러한 고형제제는 본 발명의 조성물에 적어도 하 나 이상의 부형제 예를 들면， 전분， 칼슘카보네이트 (calcium carbonate) , 수크로스 (sucrose) 또는 락토오스 ( lactose)， 젤라틴 등을 섞어 조제된다. 또한 단순한 부 형제 이외에 마그네슘 스테아레이트 탈크 같은 윤활제들도 사용된다. 경구를 위 한 액상 제제로는 현탁제， 내용액제， 유제， 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물， 리퀴드 파라핀 이외에 여러 가지 부형제， 예를 들면 습윤제， 감 미제， 방향제， 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액， 비수성용제， 현탁제， 유제， 동결건조 제제, 좌제가 포함된다. 비수성용제， 현탁제로는 프로필렌글리콜 (propylene glycol ) , 폴리에틸렌 글리콜， 올리브 오일과 같은 식물성 기름， 에틸을레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있 다. 좌제의 기제로는 위템솔 (wi tepsol ) , 마크로골, 트원 (tween) 61， 카카오지， 라 우린지， 글리세로젤라틴 둥이 사용될 수 있다. The pharmaceutical compositions according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. And in the form of sterile injectable solutions. Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sudrose, solbi, manny, xylly, erythri, malty, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyridone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil Can be mentioned. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually prepared. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid form preparations may include at least one excipient such as starch, calcium carbonate, water, or the like. Prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple brothers, lubricants such as magnesium stearate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl acrylate can be used. Suppository bases include wi tepsol, macrogol, tween 61, cacao butter, laurige and glycerogelatin.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여 (예를 들어， 정맥 내， 피하， 복강 내 또는 국소에 적용)할 수 있으며， 투여량 은 환자의 상태， 체중， 연령， 성별， 식이, 배설률， 질환의 중증도， 약물형태， 투여 시간， 투여방법， 투여켱로 및 투여기간 등에 따라 그 범위가 다양하다. 1일 투여 량은 본 발명에 따른 추출물， 분획물을 동결건조하였을 때의 양으로 0.0001 nig/kg 내지 500 nig/kg , 바람직하게는 0.001 nig/ kg 내지 100 nig/kg 이며， 필요에 따라 일 일 1회 내지 수회로 나누어 투여할 수 있다. The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage may be based on the patient's condition, weight, age, The range varies depending on sex, diet, excretion rate, severity of disease, drug type, administration time, administration method, administration period and administration period. The daily dose is 0.0001 nig / kg in the amount of lyophilized extracts and fractions according to the invention. To 500 nig / kg, preferably 0.001 nig / kg to 100 nig / kg, and may be administered once to several times a day if necessary.
본 발명의 약학적 조성물은 단독으로， 또는 수술， 방사선 치료， 호르몬 치 료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. 또한， 본 발명은 오미자추출물의 에틸아세테이트 분획물을 유효성분으로 포 함하는 염증 질환의 예방 및 개선용 건강식품을 제공한다.  The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers. In addition, the present invention provides a health food for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
본 발명의 오미자추출물의 에틸아세테이트 분획물은 HepG2 세포에서 IL-6에 의한 STAT3의 인산화를 억제하고， in vivo에서 zymosan A에 의해 유도되는 염증 정 도를 억제함으로써， 염증 질환 예방 및 개선용 건강식품의 유효성분으로 유용하게 사용될 수 있다.  Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits the phosphorylation of STAT3 by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo. It can be usefully used as an active ingredient.
본 발명의 건강식품은 오미자추출물의 에틸아세테이트 분획물을 그대로 첨 가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고， 통상적인 방법에 따라 적절하게 사용될 수 있다.  The health food of the present invention may be added with ethyl acetate fraction of Schizandra chinensis extract as it is, or may be used with other food or food ingredients, and may be appropriately used according to a conventional method.
상기 건강식품의 종류에는 특별한 제한은 없다. 오미자추출물의 에틸아세 테이트 분획물을 첨가할 수 있는 식품의 예로는 육류， 소시지 빵, 초콜릿， 캔디류， 스넥류， 과자류， 피자， 라면， 기타 면류, ¾류， 아이스크림류를 포함한 낙농제품， 각종 스프， 음료수， 차， 드링크제， 알코올음료 및 비타민 복합제 등이 있으며， 통 상적인 의미에서의 건강식품을 모두 포함한다.  There is no particular limitation on the kind of the health food. Examples of foods to which the ethyl acetate fraction of Schizandra chinensis extract can be added include meat, sausage bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, ¾, ice cream, including various kinds of soups, drinks , Teas, drinks, alcoholic beverages and vitamin complexes, and includes all healthy foods in the general sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여라 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로사 함유할 수 있다. 상기 천연 탄수화물은 포도 당， 과당과 같은 모노사카라이드, 말토스， 슈크로스와 같은 디사카라이드， 및 덱스 트린, 사이클로덱스트린과 같은 폴리사카라이드， 자일리를， 소르비를， 에리트리를 등의 당알콜이다. 감미제로서는 타우마틴， 스테비아 추출물과 같은 천연 감미제나, 丄 ύ 사카린， 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화 물의 비율은 본 발명의 조성물 100 당 일반적으로 약 0.01~0.04 g, 바람직하게는 약 0.02 ~ 0.Q3 g 이다. 상기 외에 본 발명의 갼강식품은 여러 가지 영양제, 비타 민， 전해질， 풍미제, 착색제, 펙트산 및 그의 염， 알긴산 및 그의 염， 유기산, 보 호성 콜로이드 증점제， pH 조절제， 안정화제， 방부제， 글리세린， 알코올， 탄산음료 에 사용되는 탄산화제 등올 함유할 수 있다. 그 밖에 천연 과일주스， 과일주스 음 료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으 로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01 ~ 0.1 충량부의 범위에서 선택되는 것이 일반 적이다. 또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질환의 예방 및 개선용 화장료 조성물을 제공한다. 또한， 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 화장료 조성 물을 제공한다. The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. The natural carbohydrates include sugars such as glucose, monosaccharides such as fructose, malsaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. to be. As sweeteners, natural sweeteners such as taumartin, stevia extract, Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally from about 0.01 to 0.04 g, preferably from about 0.02 to 0.03 g, per 100 compositions of the present invention. In addition to the above, the health food of the present invention is a variety of nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, It may contain an alcoholic agent such as alcohol and carbonated drinks. In addition it may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. The present invention also provides a cosmetic composition for the prevention and improvement of inflammatory diseases comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient. In addition, it provides a cosmetic composition for wound healing comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
본 발명의 오미자추출물의 에틸아세테이트 분획물은 HepG2 세포에서 IL-6에 의한 STAT3의 인산화를 억제하고, in vivo에서 zymosan A에 의해 유도되는 염증 정 도를 억제함으로써， 염증 질환 예방 및 개선용 화장료 조성물의 유효성분으로 유용 하게 사용될 수 있다. 또한, 상처치유용 화장료 조성물의 유효성분으로 유용하게 사용될 수 있다.  Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits phosphorylation of STAT3 by IL-6 in HepG2 cells and inhibits the degree of inflammation induced by zymosan A in vivo, thereby preventing and improving inflammatory diseases. It can be usefully used as an active ingredient. In addition, it can be usefully used as an active ingredient of the cosmetic composition for wound healing.
상기 화장료조성물은 예를 들면 용액， 겔, 고체 또는 반죽 무수 생성물， 수상에 유상을 분산시켜 얻은 에멀견， 현탁액， 마이크로에멀젼， 마이크로캡슐， 미 세과립구 또는 이은형 (리포좀)， 비이온형의 소낭 분산제의 형태， 크림， 스킨， 로션, 파우더， 연고， 스프레이 또는 콘실 스틱의 형태로 제공될 수 있다. 또한， 포말 ( foam)의 형태 또는 압축된 추진제를 더 함유한 에어로졸 조성물의 형태로도 제조 丄 «3 될 수 있다. The cosmetic composition may be, for example, an emulsion, suspension, microemulsion, microcapsules, fine granulocytes or dihydrate (liposomes), or non-vesicle vesicle dispersant obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, or water phase. It can be provided in the form of cream, skin, lotion, powder, ointment, spray or cone stick. It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant. 丄 «3
또한， 상기 화장료 조성물은 본 발명의 오미자추출물의 에틸아세테이트 분 획물에 추가로 지방 물질 , 유기 용매 , 용해제， 농축제 및 겔화제， 연화제， 항산화 제， 현탁화제， 안정화제， 발포제 ( foaming agent ) , 방향제， 계면활성제， 물， 이온형 또는 비이온형 유화제， 충전제, 금속이온봉쇄제 및 킬레이트화제， 보존제， 비타민， 차단제， 습윤화제， 필수 오일, 염료， 안료， 친수성 또는 친유성 활성제， 지질 소낭 또는 화장품에 통상적으로 사용되는 임의의 다른 성분과 같은 화장품 분야에서 통 상적으로 사용되는 보조제를 함유할 수 있다.  In addition, the cosmetic composition, in addition to the ethyl acetate fraction of the Schizandra chinensis extract of the present invention, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, Fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or It may contain auxiliaries commonly used in the cosmetic field, such as any other ingredients commonly used in cosmetics.
본 발명의 오미자추출물의 에틸아세테이트 분획물을 함유하는 화장료 조성 물에 있어서， 통상적으로 함유되는 화장료 조성물에 본 발명의 분획물이 1 내지 15 중량 %， 바람직하게는 2 내지 10 중량 %의 양으로 첨가될 수 있다. 또한， 본 발명은 약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 염증 질환에 걸린 개체에 투여하는 단계를 포함하는 염증 질환 치료방법 을 제공한다.  In the cosmetic composition containing the ethyl acetate fraction of the Schizandra chinensis extract of the present invention, the fraction of the present invention may be added in an amount of 1 to 15% by weight, preferably 2 to 10% by weight, to the cosmetic composition which is usually contained. have. The present invention also provides a method for treating inflammatory disease, comprising administering a pharmaceutically effective amount of ethyl acetate fraction of Schizandra chinensis extract to an individual suffering from an inflammatory disease.
또한， 본 발명은 약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 개체에 투여하는 단계를 포함하는 염증 질환 예방방법을 제공한다.  The present invention also provides a method for preventing inflammatory disease, comprising administering to the subject an ethyl acetate fraction of a pharmaceutically effective amount of Schisandra chinensis extract.
상기 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜 또 는 위험 비율로 잘환을 치료하기에 층분한 양을 의미하며 , 이는 개체의 질환의 종 류， 중증도, 약물의 활성 약물에 대한 민감도， 투여시간， 투여 경로 및 배출비율， 치료기간， 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요 소에 따라 결정될 수 있다.  The pharmaceutically effective amount refers to an amount sufficient to treat Jalhwan at a reasonable benefit or risk ratio applicable to medical treatment, which means the type, severity, sensitivity of the drug to the active drug, and administration time. And route of administration and rate of release, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.
본 발명의 오미자 추출물의 에틸아세테이트 분획물은 HepG2 세포에서 IL-6 에 의한 STAT3의 인산화를 억제하고， in vivo에서 zymosan A에 의해 유도되는 염증 정도를 억제함으로쎄 염증 질환에 대한 강력한 효과를 나타내므로， 염증 질환 예 방 및 치료방법에 유용하게 사용될 수 있다. 또한， 본 발명은 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유용 피부외용제 조성물을 제공한다. Ethyl acetate fraction of Schizandra chinensis extract of the present invention inhibits the phosphorylation of STAT3 by IL-6 in HepG2 cells and exhibits a potent effect on inflammatory diseases by inhibiting the degree of inflammation induced by zymosan A in vivo. Inflammatory disease example It can be usefully used in rooms and treatment methods. In addition, the present invention provides a skin external preparation composition for wound healing, comprising ethyl acetate fraction of Schizandra chinensis extract as an active ingredient.
본 발명의 오미자 추출물의 에틸아세테아트 분획물을 피부 외용제로 사용하 는 경우， 추가로 지방물질， 유기 용매， 용해제， 농축제 및 겔화제， 연화제， 항산 화제， 현탁화제， 안정화제， 발포제 ( foaming agent ) , 방향제， 계면활성제， 물， 이은 형 또는 비이온형 유화제， 충전제， 금속이온봉쇄제 및 킬레이트화제， 보존제， 비타 민， 차단제, 습윤화제， 필수 오일， 염료， 안료， 친수성 또는 친유성 활성제， 지질 소낭 또는 피부용 외용제에 통상적으로 사용되는 임의의 다른 성분과 같은 피부 과 학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 또한， 상기 성분들은 피부 과학 분야에서 일반적으로 사용되는 양으로 도입될 수 있다.  When ethyl acetate extract of Schizandra chinensis extract of the present invention is used as an external preparation for skin, it is additionally used for fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers and foaming agents. agent), fragrances, surfactants, water, di- or nonionic emulsifiers, fillers, metal-ion blockers and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents And adjuvant commonly used in the field of dermatology, such as lipid vesicles or any other ingredients conventionally used for external preparations for skin. In addition, the ingredients can be introduced in amounts generally used in the field of dermatology.
【유리한 효과] Advantageous Effects
본 발명에 따른 오미자 추출물의 에틸아세테이트 분획물은 다른 분획물과 달리 IL-6에 의해 유도되는 STAT3 경로의 활성화를 억제하는 효과가 있으므로， STAT3 경로와 관련된 다양한 염증 질환의 예방 및 치료용 약학적 조성물 또는 상처 치유용 약학적 조성물로 유용하게 사용할 수 있다. 【도면의 간단한 설명】  Ethyl acetate fraction of Schizandra chinensis extract according to the present invention, unlike other fractions have the effect of inhibiting the activation of the STAT3 pathway induced by IL-6, pharmaceutical compositions or wounds for the prevention and treatment of various inflammatory diseases associated with the STAT3 pathway It can be usefully used as a pharmaceutical composition for healing. [Brief Description of Drawings]
도 1은 오미자 추출물의 물 분획물， 핵산 분획물 및 에틸아세테이트 분획물 을 전처리시 IL— 6 처리에 의한 STAT3 단백질의 인산화를 나타낸 도이다.  1 is a diagram showing the phosphorylation of STAT3 protein by IL-6 treatment when water fraction, nucleic acid fraction and ethyl acetate fraction of Schizandra chinensis extract are pretreated.
도 2는 오미자 추출물의 에틸아세테이트 분획물 처리시， LPS에 의해 유도된 NO의 생성정도를 나타낸 도이다.  Figure 2 is a diagram showing the production of NO induced by LPS when the ethyl acetate fraction of Schizandra chinensis extract.
도 3은 오미자 추출물의 에틸아세테이트 분획물 처리시， 슬관절의 림프절의 l r Figure 3 is the treatment of ethyl acetate fraction of Schizandra chinensis extract, lr
15 크기를 나타낸 도이다. ,  Figure 15 shows the size. ,
도 4는 오미자 추출물의 에틸아세테이트 분획물 처리시， 실험동물와몸무게 의 변화를 보여주는 도이다.  Figure 4 is a diagram showing the change in weight and experimental animals when treated with ethyl acetate fraction of Schizandra chinensis extract.
도 5는 오미자 추출물의 에틸아세테이트 분획물 처리시， 발바닥의 부종 경 감 정도를 보여주는 도이다.  Figure 5 is a diagram showing the degree of edema relief of the sole when treating the ethyl acetate fraction of Schizandra chinensis extract.
도 6은 비교군， Zymosan처리군， 오미자추출물의 에틸아세테아트 분획물 처 리군의 왼발과 오른발 발바닥 두께의 평균값 차아를 나타낸 도이다.  6 is a diagram showing the average difference between the thickness of the left foot and the right foot of the comparative group, Zymosan treatment group, ethylaceteart fraction treatment group of Schisandra chinensis extract.
도 7은 오미자 추출물의 에틸아세테이트 분획물 처리에 의한 발바닥의 부종 경감 정도를 보여주는 사진이다.  Figure 7 is a photograph showing the degree of edema relief of the sole by treatment of ethyl acetate fraction of Schizandra chinensis extract.
[발명의 실시를 위한 최선의 형태] Best Mode for Implementation of the Invention
이하， 본 발명을 실시예, 실험예 '및 제조예에 의해 상세히 설명한다. Hereinafter, the present invention will be described in detail by Examples, Experimental Examples ' and Preparation Examples.
단 하기의 실시예, 실험예 및 제조예는 본 발명을 예시하는 것일 뿐， 본 발 명의 내용이 하기의 실시예， 실험예 및 제조예에 한정되는 것은 아니다.  However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples, Experimental Examples, and Preparation Examples.
<실시예 1>오미자추출물의 에틸아세테이트분획물의 제조 Example 1 Preparation of Ethyl Acetate Fraction of Schizandra chinensis Extract
오미자를 대전 약령시장에서 구입， 오미자열매 1kg을 물로 세척하고 상은에 서 건조시킨 후 에탄올을 1 : 1의 중량비로 흔합하여 4~5시간 동안 교반한 후 여지로 여과하고 저온 감압하에 에틸에테르를 첨가하면서 조심스럽게 농축하여 조추출물을 50. 1g을 얻었다. 이 조추출물을 상온에서 증류수 1리터에 녹이고 핵산 1리터를 넣 어 분액여두에 넣고 격렬히 흔든 후， 액이 분리되면 윗층인 핵산 분획물을 나누어 분리하여 증발건고시켰다. 남은 여벡에 에틸아세테이트를 1리터를 넣어 흔화 후 층이 분리되면 에틸아세테이트층을 분리하였고， 같은 방법으로 3회 에틸아세테이트 로 추출하여 그 분획물을 얻은 후 감압건조시켜 밀봉하여 극저온 넁장고에 보관하 며 실험에 사용하였다. <비교예 1>오미자추출물의 물 분획물의 제조 Buy Schisandra chinensis at Daejeon Yangnyeong Market, wash 1 kg of Schisandra chinensis with water, dry it over silver, and mix ethanol at a weight ratio of 1: 1, stir for 4 ~ 5 hours, filter it with filtrate, and add ethyl ether under reduced pressure. Carefully concentrated to give 50. 1g of crude extract. The crude extract was dissolved in 1 liter of distilled water at room temperature, 1 liter of nucleic acid was added to the aliquot, and the mixture was shaken vigorously. When the liquid was separated, the nucleic acid fraction of the upper layer was separated and evaporated to dryness. 1 liter of ethyl acetate was added to the remaining vaccum, followed by shaking to separate the ethyl acetate layer. The mixture was extracted three times with ethyl acetate in the same way. The fractions were obtained, dried under reduced pressure, sealed and stored in cryogenic storage. It was used for the experiment. Comparative Example 1 Preparation of Water Fraction of Schizandra chinensis Extract
상기 <실시예 1>과 같은 방법에서， 오미자 추출물의 물 분획물을 수득하였 다.  In the same manner as in <Example 1>, a water fraction of Schizandra chinensis extract was obtained.
<비교예 2>오미자추출물의 핵산분획물의 제조 Comparative Example 2 Preparation of Nucleic Acid Fractions of Schisandra chinensis Extract
상기 <실시예 1>과 같은 방법에서， 오미자 추출물의 핵산 분획물을 수득하 였다. , <실험예 1> IL-6에 의한 STAT3유전자의 활성 억제 확인  In the same manner as in <Example 1>, a nucleic acid fraction of Schisandra chinensis extract was obtained. Experimental Example 1 Confirmation of Inhibition of STAT3 Gene Activity by IL-6
오미자 추출물로부터 각 분획물이 염증 반웅에 중요한 STAT3 단백질의 활성 화에 미치는 효과를 확인하기 위하여， 웨스턴 블롯을 수행하였다.  Western blots were performed to determine the effect of each fraction from the Schizandra chinensis extract on the activation of STAT3 protein, which is important for inflammatory reactions.
HepG2 세포를 6 웰 플레이트에 5X104 세포 /웰로 분주， 전체 웰면적의 80% 정도까지 자랐을 때， 무혈청 배지로 교환하고 다시 6시간 후에 상기 오미자 에틸아 세테이트 분획물 사료를 여러 농도로 1시간 전처리하였다. 이후 20 ng/mi IL_6를 10분간 처리하고， 세포를 수거하여 단백질을 추출하였다. 구체적으로， 용균 완층 액 (20 mM Tris-HCl, pH 8， 137 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1 mM Na3V04, 2 mM EDTA, 1 mM PMSF, 20 mM leupeptin, 20 nig/ mi aprotonin)을 사용하여 세포를 용해시킨 후， 원심분리 (13,000xg, 15분)하여 그 상등액을 용해질로 얻었다. 단백질의 농도는 Bio-Rad DC 단백질 분석 키트를 이용하여 정량하였고， 10% SDS— PAGE 겔 (gel)을 이용하여 분리한 후， PVDF membraneCWeatran S, pore size 0.2 urn) 으로 전달시켰다. 5% 탈지분유 (50 mM Tri-HCl, pH 7.6， 150 mM NaCl, 0.2 % Tween-20, 5% skim milk)로 4°C에서 12시간 블로킹하고 T-TBS(50 mM Tri-HCl, pH 7.6, 150 mM NaCl, 0.2 % Tween-20)로 5번 세척한 후 일차항체로 p-STAT3 (1:1000 희석: Santa Cruzu biotechnology) 항체를, 이차항체로 HRP-접합의 항 -토끼 항체 (1:5000 희석： Calbiochem)¾ 사용하였다. When HepG2 cells were divided into 6 well plates at 5 × 10 4 cells / well and grown to about 80% of the total well area, the cells were exchanged with serum-free medium, and after 6 hours, the Schisandra chinensis acetate feed was pretreated at various concentrations for 1 hour. . Thereafter, 20 ng / mi IL_6 was treated for 10 minutes, and cells were collected to extract proteins. Specifically, lysate complete solution (20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3V04, 2 mM EDTA, 1 mM PMSF, 20 mM leupeptin, 20 nig / The cells were lysed using mi aprotonin, and then centrifuged (13,000xg, 15 minutes) to obtain the supernatant as lysate. Protein concentration was quantified using a Bio-Rad DC protein analysis kit, separated using a 10% SDS—PAGE gel, and then transferred to PVDF membraneCWeatran S, pore size 0.2 urn). Block 5 hours at 4 ° C with 5% skim milk powder (50 mM Tri-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20, 5% skim milk) and T-TBS (50 mM Tri-HCl, pH 7.6) , 150 mM NaCl, 0.2% Tween-20) and then p-STAT3 (1: 1000 dilution: Santa Cruzu biotechnology) antibody as primary antibody, HRP-conjugated anti-rabbit antibody as secondary antibody (1: 5000 dilution: Calbiochem) ¾ was used.
그 결과， 각 추출물의 분획물 중 물 분획물， 핵산 분획물과 달리 에틸아세 테이트 분획이 STAT3의 활성화를 농도 의존적으로 억제하는 것을 확인하였다 (도 1). <실험예 2> LPS에 의해 유도되는 대식세포에서의 NO생성량측정  As a result, it was confirmed that the ethyl acetate fraction inhibits the activation of STAT3 in a concentration-dependent manner, unlike the water fraction and the nucleic acid fraction in the fraction of each extract (FIG. 1). Experimental Example 2 Measurement of NO Production in Macrophages Induced by LPS
오미자 추출물의 에틸아세테이트 분획물이 염증반웅의 중요세포인 대식세포 (macrophage)에 미치는 영향을 살펴보기 위하여, 생쥐의 macrophage에서 LPS에 의 해 유되는 NO의 생성량을 참고 문헌 (Wang S et al., J. Ethnopharmacol . , 114(3) , pp458-462, 2007)에 기재되어 있는 방법을 이용하여 측정하였다.  To investigate the effect of ethyl acetate fraction of Schizandra chinensis extract on macrophage, an important cell of inflammatory reaction, the amount of NO produced by LPS in the macrophage of mouse was investigated (Wang S et al., J.). Ethnopharmacol, 114 (3), pp458-462, 2007).
먼저 오미자 추출물의 에틸아세테이트 분획물이 세포독성을 나타내는지를 살펴보기 위하여 기존 문헌에 기재된 MTS 분석방법을 이용하였다 (Desai A et al, J Pharm Sci. 97(7) :2745-56, 2008). RAW -264.7 세포를 96 웰 플레이트에 1x104 세 포 /웰로 분주한 후， EA 추출물을 농도별 (0, 10, 50， 100,200,300 iig/ml)로 각각 18 시간 동안 처리하였다. 웰당 20 ^의 MTS 용액을 첨가하여 37°C, 5% C02 배양 기에서 4시간 동안 반웅시킨 후, 마이크로플레이트 리더 (itiicroplate reader , DYNEX, Opsys MR, USA)를 이용하여 450 nm에서 흡광도의 변화를 측정, 추출물을 처리하지 않은 대조군에 대한 세포생존율을 백분율로 표시하였다. 300 iig/ml의 농도까지는 독성이 나타나지 않았음을 확인하였고 하기의 실험을 진행하였다 (도 2).  First, the MTS assay described in the existing literature was used to examine whether the ethyl acetate fraction of Schizandra chinensis extract showed cytotoxicity (Desai A et al, J Pharm Sci. 97 (7): 2745-56, 2008). After dispensing RAW -264.7 cells into 96 well plates at 1x104 cells / well, EA extracts were treated for 18 hours at different concentrations (0, 10, 50, 100, 200, 300 iig / ml). After 4 hours of reaction at 37 ° C., 5% C02 incubator with 20 ^ MTS solution per well, change in absorbance at 450 nm was obtained using a microplate reader (DYNEX, Opsys MR, USA). Measurement, cell viability relative to the control not treated extract was expressed as a percentage. It was confirmed that no toxicity was observed up to the concentration of 300 iig / ml and the following experiment was performed (FIG. 2).
상기 실험예 1의 방법으로 배양된 RAW 264.7 세포에 상기 실시예 1에서 수 득한 오미자 추출물의 에틸아세테이트 분획물을 다양한 농도로 전처리 하고 1시간 후 100 ng/ml의 LPS를 처리하여 18시간 배양하였다. 배양액 50 ^와 같은 양의 그 리스 반웅액 (Griess Reagent)을 넣어주고 10분간 상온에서 반웅시킨 후 Automatic EL ISA reader (precision microplate reader , Molecular Devices, BI0-TEX EL800uv-PC, USA)를 이용하여 540 nm에서 흡광도를 측정하였다. 아질산 나트륨 (sodium nitrite)의 농도별 표준곡선을 이용하여 배양액 내의 N0 농도를 결정하였 다. RAW 264.7 cells cultured by the method of Experimental Example 1 was pretreated with ethyl acetate fraction of the Schizandra chinensis extract obtained in Example 1 at various concentrations, and incubated for 18 hours by treatment with 100 ng / ml LPS after 1 hour. Add Grease Reagent in the same amount as the culture medium 50 ^ and react at room temperature for 10 minutes, and then use 540 using an Automatic EL ISA reader (precision microplate reader, Molecular Devices, BI0-TEX EL800uv-PC, USA). Absorbance was measured at nm. The concentration of N0 in the culture medium was determined using the standard curve of concentration of sodium nitrite. All.
그 결과， 오미자열매 에틸아세테이트 추출물올 30 u g/ 의 농도를 1시간 동안 전 처리하였을 때， LPS-유도된 NO의 생성 감소효과를 확인할 수 있었다 (도 2) . <실험예 3> In vivo에서 오미자추출물의 에틸아세테이트 분획물의 염증 반웅 억제 효과 분석  As a result, when pre-treatment of 30 u g / concentration of Schizandra ethyl acetate extractol for 1 hour, it was confirmed that the effect of reducing the production of LPS-induced NO (Fig. 2). Experimental Example 3 Inhibition of Inflammatory Response of Ethyl Acetate Fraction from Schizandrae Fructus Extract in Vivo
오미자 추출물의 에틸아세테이트 분획물이 염증 치료활성을 가지는지 확인 하기 위하여， 생쥐에서 염증을 유발하고 그 억제 정도를 조사하였다. C57BL/6 마 우스를 증앙실험동물에서 입수하여 1주간 순화시킨 후 실험에 사용하였다.  In order to determine whether the ethyl acetate fraction of Schisandra chinensis extract had anti-inflammatory activity, the inflammation was induced in mice and the degree of inhibition was investigated. C57BL / 6 mice were obtained from augmented experimental animals, purified for one week, and used for experiments.
염증유발을 위하여 Saccharomyces cerevi ci ae로부터 분리된 Zymosan Zymosan Isolated from Saccharomyces cerevi ci ae for Inflammation
A(Sigma-Aldr ich, St . Loui s , MO, USA)를 3 조제하여 50 를 오른쪽 발에 주사하였다. 실험군에는 오미자 추출물의 에틸아세테이트 분획물을 30 mg/kg로 오 른쪽 발에 매일 오후 4시， 하루에 한 번씩 투여하였고， 대조군으로는 아무것도 처 리하지 않거나， 상기 Zymosan A만 투여하였다. 1 2 3 4, 5일에 체중을 측정하 였으며， 그 결과를 도 4에 나타내었다. Three A (Sigma-Aldr ich, St. Loui s, Mo., USA) were prepared and 50 was injected into the right foot. In the experimental group, the ethyl acetate fraction of Schisandra chinensis extract was administered to the right foot once every day at 4 pm daily, at 4 pm, and none was treated as a control, or only Zymosan A was administered. Weight was measured on day 1 2 3 4, 5, and the results are shown in FIG.
그 결과， 도 4에 나타난 바와 같이， 본 발명와오미자 추출물의 에틸아세테 이트 분획물 처리에 의한 체중감소가 이루어지지 않았으므로， 부작용이 없는 것을 확인하였다.  As a result, as shown in Figure 4, the weight loss by the ethyl acetate fraction treatment of the present invention and Schisandra chinensis extract was not made, it was confirmed that there are no side effects.
또한， 상기와 같이 물질을 처리한 후 슬관절 림프절의 크기를 조사하였다. 그 결과 도 3에 나타난 바와 같이， 오미자 추출물의 에틸아세테이트 분획물은 zymosan A에 의한 슬관절 림프절의 비대를 억제함을 확인할 수 있었다. . 또한， 상기와 같이 물질을 처리한 후 매일 발 두께를 측정하였다.  In addition, the size of the knee joint was examined after treatment with the material as described above. As a result, as shown in Figure 3, the ethyl acetate fraction of Schisandra chinensis extract was confirmed to inhibit the hypertrophy of the knee joint lymph nodes by zymosan A. . In addition, the foot thickness was measured daily after treating the material as described above.
그 결과 도 5에 나타난 바와 같이 Zymosan A에 꾀해 발 두께가 18.7 26.7 , 26.4 25.4 24.5 23.6 隱였던 것이 오미자 추출물의 에틸아세테이트 분획물을 처 리한 경우 18.5 24.8 , 24.6 23. 1 , 21.7, 21.7 mm로 발의 부종이 경감된 것을 확 인하였다 (도 5) . 아무것도 처리하지 않은 군, Zymosan A 처리군， Zymosan A와 오 미자 추출물의 에틸아세테이트 분획물을 처리한 군의 외발과 오른발과 평균값 차이 는 1.0 , 29.5 , 18.5 隱로 나타났으며 (도 6)， 발의 실제 사진에서도 그 차이를 확인 할 수 있었다 (도 7) . As a result, as shown in FIG. 5, the foot thickness was 18.7 26.7, 26.4 25.4 24.5 23.6 해, and treated with ethyl acetate fraction of Schisandra chinensis extract 18.5 24.8, 24.6 23. 1, 21.7, 21.7 mm I confirm that this was reduced (FIG. 5). The average difference between the paw and right foot of the group treated with nothing, Zymosan A treated group, the group treated with ethyl acetate fraction of Zymosan A and Schisandra chinensis extract was 1.0, 29.5, 18.5 隱 (FIG. 6), The difference could be seen in the picture (Fig. 7).
상기와 같은 결과를 통해， 본 발명의 오미자추출물의 에틸아세테이트 분획 물은 물 및 핵산 분획물과 달리 HepG2 세포에서 IL-6에 의한 STAT3의 인산화를 억 제하고, in vivo에서 zymosan A에 의해 유도되는 염증 정도를 억제함으로써 , 염증 질환 예방 및 치료용 조성물 또는 상처치유용 조성물의 유효성분으로 유용하게 사 용될 수 있다.  Through the above results, the ethyl acetate fraction of Schizandra chinensis extract of the present invention, unlike water and nucleic acid fraction, inhibits phosphorylation of STAT3 by IL-6 in HepG2 cells, and inflammation induced by zymosan A in vivo. By suppressing the degree, it can be usefully used as an active ingredient for the composition for preventing and treating inflammatory diseases or for wound healing.
<제조예 1> 약학적 제제의 제조 Preparation Example 1 Preparation of Pharmaceutical Formulation
<1-1>산제의 제조  <1-1> Preparation of powder
본 발명의 오미자 추출물의 에틸아세테이트 분획물 2 mg  2 mg of ethyl acetate fraction of Schizandra chinensis extract of the present invention
유당 1 g  1 g lactose
상기의 성분을 흔합하고 기밀포에 층진하여 산제를 제조하몄다.  The above ingredients were mixed and layered in an airtight cloth to prepare a powder.
<1-2> 정제의 제조 <1-2> Preparation of Tablet
본 발명의 오미자 추출물의 에틸아세테이트 분획물 100 nig  100 nig of ethyl acetate fraction of Schizandra chinensis extract of the present invention
옥수수전분 100 mg  Corn starch 100 mg
유 당 100 nig  100 nig lactose
스테아린산 마그네슘 2 nig  2 nig magnesium stearate
상기의 성분을 흔합한 후， 통상의 정제의 제조방법에 따라서 타정하여 정제 를 제조하였다. <1-3> 캡슐제의 제조 본 발명의 오미자 추출물의 에틸아세테이트 분획물 100 mg After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets. <1-3> Preparation of Capsule 100 mg of ethyl acetate fraction of Schizandra chinensis extract of the present invention
옥수수전분 100 nig  Corn starch 100 nig
유 당 100 mg .  Lactose 100 mg.
스테아린산 마그네슘 2 mg  2 mg magnesium stearate
상기의 성분을 흔합한 후， 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐 에 충전하여 캡슐제를 제조하였다.  After mixing the above components, it was filled into gelatin capsules according to the conventional method for producing a capsule to prepare a capsule.
<1-4>환의 제조 <1-4> Preparation of the ring
본 발명의 오미자 추출물의 에틸아세테이트 분획물 1 mg  1 mg of ethyl acetate fraction of Schizandra chinensis extract of the present invention
유 당 1.5 g  1.5 g lactose
글리세린 1 g  1 g of glycerin
자일리를 0.5 g  0.5 g of Xili
상기의 성분을 흔합한 후， 통상의 방법에 따라 1 환 당 4 g이 되도록 제조 하였다. After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.
<1-5>과립의 제조 <1-5> Preparation of granules
본 발명의 오미자 추출물의 에틸아세테이트 분획물 150 nig  150 nig of ethyl acetate fraction of Schizandra chinensis extract of the present invention
대두 추출물 50 nig  Soybean Extract 50 nig
포도당 200 mg  Glucose 200 mg
전분 600 mg  Starch 600 mg
상기의 성분을 흔합한 후， 30% 에탄올 100 mg을 첨가하여 섭씨 60°C에서 건 조하여 과립을 형성한 후 포에 충진 하였다. After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C to form granules, and then filled into fabrics.
<제조예 2>식품의 제조 Preparation Example 2 Preparation of Food
<2-1>밀가루 식품의 제조 본 발명의 오미자 추출물의 에틸아세테이트 분획물 0.5~5.0 중량부를 밀가 루에 첨가하고， 이 흔합물을 이용하여 빵， 케이크， 쿠키， 크래커 및 면류를 제조하 였다. <2-1> Preparation of flour food 0.5-5.0 parts by weight of ethyl acetate fraction of Schizandra chinensis extract of the present invention was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.
<2-2>스프 및 육즙 (gravies)의 제조 <2-2> Preparation of Soup and Gravy
본 발명의 오미자 추출물의 에틸아세테이트 분획물의 0. 1~5.0 중량부를 스 프 및 육즙에 첨가하여 건강 증진용 육가공 제품， 면류의 수프 및 육즙을 제조하였 다ᅳ  0.1-5.0 parts by weight of the ethyl acetate fraction of Schizandra chinensis extract of the present invention was added to soup and broth to prepare health-promoting meat products, noodles soup and broth.
<3-3>그라운드 비프 (ground beef)의 제조 <3-3> Preparation of ground beef
본 발명의 오미자 추출물의 에틸아세테이트 분획물 10 중량부를 그라운드 비프에 첨가하여 건강 증진용 그라운드 비프를 제조하였다.  10 parts by weight of the ethyl acetate fraction of the Schizandra chinensis extract of the present invention was added to the ground beef to prepare a ground beef for health promotion.
<3-4>유제품 (dairy products)의 제조 <3-4> Manufacture of dairy products
본 발명의 오미자 추출물의 에틸아세테이트 분획물 5~10 중량부를 우유에 첨가하고， 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조 하였다.  5 to 10 parts by weight of ethyl acetate fraction of Schizandra chinensis extract of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.
<3-5>선식의 제조 <3-5> Preparation of wire
현미， 보리， 찹쌀， 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전 한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.  Brown rice, barley, glutinous rice, yulmu was alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.
검정콩, 검정깨， 들깨도 공지의 방법으로쪄서 건조시킨 것을 배전한 후 분 쇄기로 입도 60 메쉬의 분말로 제조하였다.  Black beans, black sesame seeds, and perilla were also roasted by a known method and then roasted to prepare a powder having a particle size of 60 mesh using a mill.
본 발명의 오미자 추출물의 에틸아세테이트 분획물을 진공 농축기에서 감압 농축하고， 분무， 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60 메쉬로 분쇄하여 건조분말을 얻었다. The ethyl acetate fraction of Schizandra chinensis extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and a hot air dryer, and then dried to a particle size of 60 mesh by a grinder. Grinding to obtain a dry powder.
상기에서 제조한 곡물류ᅳ 종실류 및 오미자 추출물의 에틸아세테이트 분획 물을 다음의 비율로 배합하여 제조하였다.  The ethyl acetate fractions of the grains ᅳ seeds and Schisandra chinensis extracts prepared above were prepared by combining the following ratios.
곡물류 (현미 30 중량부， 율무 15 중량부， 보리 20 중량부)，  Cereals (30 parts by weight brown rice, 15 parts by weight of radish, 20 parts by weight of barley) ，
종실류 (들깨 7 중량부， 검정콩 8 중량부， 검정깨 7 중량부)，  Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),
오미자 추출물의 에틸아세테이트 분획물 (3 중량부)，  Ethyl acetate fraction of Schizandra chinensis extract (3 parts by weight) ，
영지 (0.5 중량부)，  Manor (0.5 parts by weight) ，
지황 (0.5 중량부) <제조예 3>음료의 제조  Foxglove (0.5 parts by weight) <Preparation Example 3> Preparation of the beverage
<3-1> 건강음료의 제조  <3-1> Preparation of health drink
액상과당 (0.5 %) , 올리고당 (2 ) , 설탕 (2 %) , 식염 (0.5 %) , 물 (75 %)과 같 은 부재료와 본' 발명인 오미자 추출물의 에틸아세테이트 분획물 5 g을 균질하게 배 합하여 순간 살균을 한 후 이를 유리병， 패트병 등 소포장 용기에 포장하여 제조하 였다. Liquid fructose (0.5%), oligosaccharide (2), sugar (2%), salt (0.5%), combined homogeneously times the ethyl acetate fraction 5 g of the present "inventors Schizandra extract and the sub material, such as water (75%) After instant sterilization it was prepared by packaging in small packaging containers such as glass bottles and plastic bottles.
<3-2> 야채 주스의 제조 <3-2> Preparation of Vegetable Juice
본 발명의 오미자 추출물의 에틸아세테이트 분획물 5 g을 토마토 또는 당근 주스 1 , 000 m£에 가하여 야채 주스를 제조하였다.  Vegetable juice was prepared by adding 5 g of ethyl acetate fraction of Schizandra chinensis extract to tomato or carrot juice 1,000 m £.
<3-3>과일 주스의 제조 <3-3> Preparation of fruit juice
본 발명의 오미자 추출물의 에틸아세테이트 분획물 1 g을 사과 또는 포도 주스 1 , 000 에 가하여 과일 주스를 제조하였다. <제조예 4>화장품의 제조 본 발명와 오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 함유하 는 염증 질환의 예방 및 개선용 화장품을 제조할 수 있다. 본 발명자들은 상기 화 장품으로 영양화장수， 크림， 에센스 등의 유화 제형의 화장품 및 유연화장수 등의 가용화 제형의 화장품을 제조하였다. Fruit juice was prepared by adding 1 g of ethyl acetate fraction of Schizandra chinensis extract to 1,000 apple or grape juices. Preparation Example 4 Preparation of Cosmetics The present invention can be prepared cosmetics for the prevention and improvement of inflammatory diseases containing ethyl acetate fraction of Schizandra chinensis extract as an active ingredient. The present inventors prepared cosmetics of solubilized formulations, such as cosmetics of the emulsified formulations such as nutrient cosmetics, creams, essences and soft cosmetics as the cosmetics.
<4-1>유화 제형의 화장품 제조 <4-1> Cosmetic preparation of emulsified formulation
하기 [표 1]에 기재된 조성으로 유화제형의 화장품을 제조하였다. 제조 방 법은 하기와 같다.  To the cosmetic composition of the emulsifier type was prepared in the composition shown in Table 1. The manufacturing method is as follows.
1) 1 내자 9의 원료를 흔합한 혼합물을 65 - 70 °C로 가열하였다. 1) The mixed mixture of 1 to 9 raw materials was heated to 65-70 ° C.
2) 10의 원료를 상기 단계 1)의 흔합물에 투입하였다.  2) 10 raw materials were added to the mixture of step 1).
3) 11 내지 13의 원료의 혼합물을 65 ~ 70 °C로 가열하여 완전히 용해시켰 다. 3) The mixture of raw materials of 11 to 13 was heated to 65-70 ° C and completely dissolved.
4) 상기 단계 3)을 거치면서， 상기 2)의 흔합물을 서서히 첨가하여 6 , 000 rpm에서 2 ~ 3분간 유화시켰다.  4) While passing through the step 3), the mixture of 2) was slowly added to emulsify for 2-3 minutes at 6,000 rpm.
5) 14의 원료를 소량와 물에 용해시킨 후 상기 단계 4)의 흔합물에 첨가하 고 2분간 더 유화시켰다.  5) The raw material of 14 was dissolved in a small amount and water, and then added to the mixture of step 4) and emulsified for 2 minutes.
6) 15 내지 17의 원료를 각각 정량한 후 상기 단계 5)의 흔합물을 40 °C에 서 30초간 더 유화시켰다. 6) After quantifying the raw materials of 15 to 17, the mixture of step 5) was further emulsified at 40 ° C. for 30 seconds.
7) 상기 단계 6)의 흔합물을 유화 후 탈기과정을 거쳐 25 - 35 °C로 냉각시 킴으로써 유화제형의 화장품을 제조하였다. 7) The emulsion of step 6) was emulsified and then degassed and cooled to 25-35 ° C. to prepare an emulsified cosmetic.
【표 1] [Table 1]
조성 유화제형 1 유화제형 2 유화제형 3Composition Emulsifier 1 Emulsifier 2 Emulsifier 3
1 스테아린 산 0.3 0.3 0.3 1 stearic acid 0.3 0.3 0.3
2 스테알리 알콜 0.2 0.2 0.2
Figure imgf000026_0001
2 steali alcohol 0.2 0.2 0.2
Figure imgf000026_0001
<4-2>가용화 제형의 화장품 제조 <4-2> Cosmetic preparation of solubilized formulation
하기 [표 2]에 기재된 조성으로 가용화 제형의 화장품을 제조하였다. 제조 방법은 하기와 같다.  A cosmetic of a solubilized formulation was prepared with the composition shown in Table 2 below. The manufacturing method is as follows.
1) 2 내지 6의 원료를 1의 원료 (정제수)에 넣고 아직믹서를 이용하여 용해 시켰다.  1) 2 to 6 raw materials were put into 1 raw material (purified water) and dissolved using a still mixer.
2) 8 내지 11의 원료를 7의 원료 (알코올)에 넣고 완전 용해시켰다. 2) The raw materials of 8 to 11 were placed in the raw material of 7 (alcohol) and completely dissolved.
3) 상기 단계 2)의 혼합물을 상기 단계 1)의 흔합물에 서서히 첨가하면서 가용화시켰다.
Figure imgf000027_0001
3) The mixture of step 2) was solubilized with slow addition to the mixture of step 1).
Figure imgf000027_0001

Claims

【청구의 범위】 【Scope of Claim】
【청구항 1】 , . 【Claim 1】 , .
Figure imgf000028_0001
BMLL. ) 추출물의 에틸아세테이트 분획물을 유효성분 으로 포함하는 염증 질환의 예방 및 치료용 약학적 조성물.
Figure imgf000028_0001
BMLL. ) A pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the ethyl acetate fraction of the extract as an active ingredient.
【청구항 2】 【Claim 2 】
제 1항에 있어서， 오미자 추출물의 에틸아세테이트 분획물은 STAT3의 인산화 를 억제하는 것을 특징으로 하는 염증 질환의 예방 및 치료용 약학적 조성물. The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 1, wherein the ethyl acetate fraction of Schisandra chinensis extract inhibits phosphorylation of STAT3.
[청구항 3】 [Claim 3]
제 1항에 있어서， 오미자 추출물의 에틸아세테이트 분획물은 N0(Ni tr ic Oxide)의 생성을 억제하는 것을 특징으로 하는 염증 질환의 예방 및 치료용 약학적 조성물. The pharmaceutical composition for the prevention and treatment of inflammatory diseases according to claim 1, wherein the ethyl acetate fraction of Schisandra chinensis extract inhibits the production of N0 (Ni tr ic Oxide).
[청구항 4】 [Claim 4]
제 1항에 있어서， 염증 질환은 부종, 자가면역질환， 관절염， 복막염， 건선， 천식， 다발성경화증， 알레르기, 기관지 경련， 파괴성 골질환， 감염성 질환， 퇴행성 질환， 괴사성 질환 및 염증성 치주질환으로 이루어진 군으로부터 선택되는 1종 이 상인 것을 특징으로 하는 약학적 조성물. The method of claim 1, wherein the inflammatory disease consists of edema, autoimmune disease, arthritis, peritonitis, psoriasis, asthma, multiple sclerosis, allergy, bronchospasm, destructive bone disease, infectious disease, degenerative disease, necrotic disease, and inflammatory periodontal disease. A pharmaceutical composition, characterized in that it is one or more types selected from the group.
【청구항 5】 【Claim 5】
제 1항에 있어서， 오미자 추출물은 오미자를 물， C1 내지 C2의 저급 알콜 또 는 이들의 흔합용매를 이용하여 넁침， 가열， 초음파 또는 환류 넁각으로 추출한 것 을 특징으로 하는 염증 질환의 예방 및 치료용 약학적 조성물. According to claim 1, the Schisandra chinensis extract is for the prevention and treatment of inflammatory diseases, characterized in that Schisandra chinensis is extracted by acupuncture, heating, ultrasound, or reflux using water, C1 to C2 lower alcohol, or a common solvent thereof. Pharmaceutical composition.
【청구항 6】 【Claim 6】
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질 환의 예방 및 개선용 건강식품, Health food for preventing and improving inflammatory diseases containing the ethyl acetate fraction of Schisandra chinensis extract as an active ingredient,
[청구항 7】 [Claim 7]
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 염증 질 환의 예방 및 개선용화장료 조성물. A cosmetic composition for preventing and improving inflammatory diseases comprising the ethyl acetate fraction of Schisandra chinensis extract as an active ingredient.
【청구항 8】 【Claim 8】
약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 염증 질 환에 걸린 개체에 투여하는 단계를 포함하는 염증 질환 치료방법. A method of treating an inflammatory disease comprising administering a pharmaceutically effective amount of the ethyl acetate fraction of Schisandra chinensis extract to a subject suffering from an inflammatory disease.
【청구항 9】 【Claim 9】
약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 개체에 투여하는단계를 포함하는 염증 질환 예방방법 . A method for preventing inflammatory diseases comprising administering a pharmaceutically effective amount of the ethyl acetate fraction of Schisandra chinensis extract to a subject.
【청구항 10】 【Claim 10】
염증 질환 예방 및 치료용 약학적 조성물로 사용하기 위한 오미자 추출물의 에틸아세테이트 분획물의 용도. Use of the ethyl acetate fraction of Schisandra chinensis extract for use as a pharmaceutical composition for preventing and treating inflammatory diseases.
― ―
【청구항 11】 【Claim 11】
염증 질환 예방 및 개선용 건강기능 식품으로 사용하기 위한 오미자 추출물 의 에틸아세테이트 분획물의 용도. Use of the ethyl acetate fraction of Schisandra chinensis extract for use as a health functional food for preventing and improving inflammatory diseases.
【청구항 12】 염증 질환 예방 및 개선용 화장료 조성물로 사용하기 위한 오미자 추출물의 에틸아세테이트 분획물의 용도. 【Claim 12】 Use of the ethyl acetate fraction of Schisandra chinensis extract for use as a cosmetic composition for preventing and improving inflammatory diseases.
[청구항 13】 [Claim 13]
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유 용 약학적 조성물. A pharmaceutical composition for wound healing comprising the ethyl acetate fraction of Schisandra chinensis extract as an active ingredient.
【청구항 14】 . 【Claim 14】.
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유 용 피부외용제 조성물. An external skin composition for wound healing comprising the ethyl acetate fraction of Schisandra chinensis extract as an active ingredient.
【청구항 15】 【Claim 15】
오미자 추출물의 에틸아세테이트 분획물을 유효성분으로 포함하는 상처치유 용 화장료 조성물. A cosmetic composition for wound healing comprising the ethyl acetate fraction of Schisandra chinensis extract as an active ingredient.
【청구항 16】 【Claim 16】
약학적으로 유효한 양의 오미자 추출물의 에틸아세테이트 분획물을 상처에 처리하는 단계를 포함하는 상처치유 방법 . 【청구항 17】 A wound healing method comprising treating a wound with a pharmaceutically effective amount of the ethyl acetate fraction of Schisandra chinensis extract. 【Claim 17】
상처치유용 조성물로 사용하가 위한 오미자 추출물의 에틸아세테이트 분획 물의 용도. Use of the ethyl acetate fraction of Schisandra chinensis extract for use as a wound healing composition.
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